AH diagnostics: Your Science Our Focus

Focus Western Blot AH diagnostics: Your Science – Our Focus Dedicated to helping life science and diagnostics laboratories perform their best by pr...
Author: Garey Short
2 downloads 0 Views 2MB Size
Focus

Western Blot

AH diagnostics: Your Science – Our Focus Dedicated to helping life science and diagnostics laboratories perform their best by providing innovative, high-quality products and expert knowledge.

Representing more than 35 suppliers, our application specialists are ready to help you with your next project, from assay setup, training and implementation to analysis.

We offer the most cutting edge technologies within ELISA, flow cytometry, imaging, pathology, protein analysis, qPCR, single-cell analysis and bio appliance.

We have 30 years of experience serving the Nordic Region and offices in Copenhagen, Aarhus, Oslo, Stockholm and Helsinki.

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY Western Blot Overview Western blotting is as a simple and straightforward technique for protein characterization. However, a great number of steps are required to secure optimal results, and we know it is crucial to be confident with the entire process. We have therefore published this magazine to support you in making the right Western blotting choice. Use the above workflow throughout the magazine as a guideline in your daily Western blot work. Simply identify your step of interest and become inspired by our solutions. Despite Western blotting being a widely used and simple technique, many challenges can still arise. If you come across a problem, turn to the troubleshooting section at the end of the magazine, or contact one of our trained specialists for further support.

1 Sample Preparation��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 5 1.1 Cell Lysis����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 5 1.2 Protease and Phosphatase Inhibition �������������������������������������������������������������������������������������������������������������������������������������������������� 6 1.3 Purification������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 7 1.3.1 His-Tag purification����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 7 1.3.2 FLAG-Tag Purification products������������������������������������������������������������������������������������������������������������������������������������������������������������� 8 1.3.3 GST-Tag Purification products���������������������������������������������������������������������������������������������������������������������������������������������������������������� 9 1.3.4 Myc-Tagged Protein Purification������������������������������������������������������������������������������������������������������������������������������������������������������������ 9 1.3.5 Non-Tagged protein purification����������������������������������������������������������������������������������������������������������������������������������������������������������10 1.4 Concentration Measuring���������������������������������������������������������������������������������������������������������������������������������������������������������������������12 2 Protein Separation��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 13 2.1 Protein Concentration and Buffer Exchange�������������������������������������������������������������������������������������������������������������������������������������13 2.2 Sample Loading Buffers������������������������������������������������������������������������������������������������������������������������������������������������������������������������14 2.2.1 Reducing and Non-reducing Sample Loading Buffers��������������������������������������������������������������������������������������������������������������������14 2.2.2 Real-time, In-gel Protein Stain���������������������������������������������������������������������������������������������������������������������������������������������������������������14 2.3 Gel Electrophoresis Buffers������������������������������������������������������������������������������������������������������������������������������������������������������������������15 2.4 Molecular Weight Standards����������������������������������������������������������������������������������������������������������������������������������������������������������������15 2.5 Gel Cast and Run������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������16 2.6 Protein Stain���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������16 3 Blotting��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 17 3.1 Membranes����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������17 3.2 Blotting Papers����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������18 3.3 Transfer Buffer�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������18 3.4 Protein Stain for Membranes���������������������������������������������������������������������������������������������������������������������������������������������������������������19 4 Incubation with Antibodies������������������������������������������������������������������������������������������������������������������������������������������������������������������ 20 4.1 Blocking Solution������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������20 4.2 Washing Buffer for Western Blots�������������������������������������������������������������������������������������������������������������������������������������������������������20 4.3 Direct or Indirect Methods?�����������������������������������������������������������������������������������������������������������������������������������������������������������������20 4.4 Primary Antibodies���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������21 4.5 Secondary Antibodies���������������������������������������������������������������������������������������������������������������������������������������������������������������������������21 4.6 Streptavidin Conjugates������������������������������������������������������������������������������������������������������������������������������������������������������������������������23 5 Detection������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 24 5.1 HRP Substrates����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������24 5.2 How to Choose the Right Chemistry for Your Western Blot ��������������������������������������������������������������������������������������������������������25 5.2.1 Chemiluminescence������������������������������������������������������������������������������������������������������������������������������������������������������������������������������25 5.2.2 Fluorescence�������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������25 6 Troubleshooting������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 27 6.1 Unusual/Unexpected Bands����������������������������������������������������������������������������������������������������������������������������������������������������������������27 6.2 No Bands, Faint Bands or Weak Signal����������������������������������������������������������������������������������������������������������������������������������������������28 6.3 Electrophoretic Problems����������������������������������������������������������������������������������������������������������������������������������������������������������������������28 6.4 High Background������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������29

3

4

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 1 Sample Preparation The first step in obtaining a successful Western blot is to properly prepare the starting material. Prior to sample preparation, it is of the utmost importance to acquire knowledge about the protein location, protein source, hydrophobicity of protein target, etc. Whether you are working with cultured cell lines or tissue samples, sample preparation is often a process of trial and error in which you need to choose the right lysis buffers, enzyme inhibitors and appropriate volumes. Some samples may even require mechanical disruption.

1.1 Cell Lysis Cell lysis is often performed using a buffer containing detergents and/or denaturants. The choice of buffer depends on cell location. From Santa Cruz, we offer:

Protein location

Buffer

Whole cell

NP-40, RIPA, Triton X-100

Cytoplasmic (soluble)

Tris-HCl

Cytoplasmic (cytoskeletal bound)

Tris-Triton

Nuclear, mitochondrial, membrane

RIPA, Triton X-100, NP-40

Clontech offers the mild, non-denaturing xTractor Lysis Buffer for fast and easy extraction of proteins from bacteria, yeast, mammalia, and baculo-infected cells. The product is compatible with IMAC resins for quick purification of His-tagged proteins. It takes only 10 minutes!

Cat. no.

Description

Size

635625

xTractor Buffer

500 mL

635656

xTractor Buffer

100 mL

635671

xTractor Buffer

250 mL

635623

xTractor Buffer Kit

10 extractions

5



Sample Preparation



Protein Separation

Blotting



AB Incubation



Detection

YY 1.2 Protease and Phosphatase Inhibition Cell lysis is associated with the release of proteases and phosphatases, which in turn negatively affects the Western blot. UltraCruz® Protease Inhibitor Cocktail Tablet prevents proteolysis from bacterial, yeast, mammalian and plant extracts! Available with or without EDTA. Simply dissolve the tablet in buffer or lysate.

Cat. no.

Description

Size

Sc-29130

UltraCruz Protease Inhibitor Cocktail Tablet

20 tablets

Sc-29131

UltraCruz® Protease Inhibitor Cocktail Tablet, EDTA-free

20 tablets

®

ProteoGuard™ EDTA-Free Protease Inhibitor from Clontech is a cocktail in solution. Provided in single-use tubes for ease of use.

Cat. no.

Description

Size

635672

ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail

100 uL

635673

ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail

10 x 100 uL

For phosphatase inhibitors, use the following mixes from Santa Cruz:

Cat. no.

Size

Inhibitor mix

Target

Sc-45044

1 mL

Phosphatase Inhibitor Cocktail A

Serine/threonine protein phosphatases, L-isozymes of alkaline

Sc-45045

1 vial*

Phosphatase Inhibitor Cocktail B

Tyrosine phosphatases, acid and alkaline phosphatases

Sc-45065

1 mL

Phosphatase Inhibitor Cocktail C

Alkaline phosphatases, serine/ threonine phosphatases

*reconstitute in 1 mL water

6

Looking for inhibitors that target specific groups of proteases and phosphatases? Contact us for customized solutions from Santa Cruz or Enzo.

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 1.3 Purification It can be advantageous to purify proteins by other means than lysing the cells with detergents and denaturants combined with mechanical stress and inhibitors. A large number of methods are available both for purifications prior to Western blotting studies, but also for larger scale downstream purifications. Choose your protein purification products from Takara Clontech. Whether you are working with tagged or non-tagged targets,Takara Clontech offers products for a large number of applications. The below listed categories contains their full protein purification product range.

His-tag purification products • Capturem™ His-tagged Purification Kit • His60 Ni • TALON® • Magnetic Beads FLAG-tag purification products • Magnetic DYKDDDDK Immunoprecipitation Kit • Anti-DYKDDDDK Beads GST-tag purification products • Glutathione-Superflow Resin

Phosphoprotein enrichment products • Phosphoprotein Enrichment Kit • TALON® PMAC Magnetic Phospho Enrichment Kit • Phosphopeptide Enrichment Spin Columns Glycoprotein enrichment products • Glycoprotein Enrichment Resin Antibody purification products • Thiophilic-Superflow Resin

Myc-tag purification products • c-Myc Monoclonal Antibody-Agarose Beads • Magnetic Myc Immunoprecipitation Kit

Please contact us for ordering information and further details on the specific products.

1.3.1 His-Tag purification Many conventional methods for protein purification require a lot of time and effort. Immobilized metal affinity chromatography resin columns provide high capacity, but speed and ease of use are compromised due to long equilibration and binding times, as well as slow diffusion of large molecules through the resin bed. A next-generation membrane technology utilizing spin columns, which overcomes these bottlenecks, is now available from Takara Clontech. Purify His-tagged proteins in 5-15 minutes with Capturem® His-tagged purification kit • • • •

NEW!

4 step protocol at room temperature Purification of mammalian or bacterial His-tagged proteins Compatible with both native and denaturing conditions containing DTT, β-mercaptoethanol, TCEP, EDTA or glycerol High yield and purity

7

Sample Preparation



Protein Separation





Blotting

AB Incubation

YY Equilibration: Add xTractor buffer, spin, discard flowthrough

Binding: Load cleared lysate, spin + save flowthrough, transfer column to new tube

Washing: Add wash buffer. If needed, add small amount of elution buffer to change imidazole conc. and optimize, spin, save flowthrough, transfer column to new tube

Elution: Add elution buffer, spin, elution contains purified and tagged protein

Miniprep

Maxiprep

High-throughput 96 minipreps

Cat. no.

635710

635713

635714

Yield

~ 100 µg/prep

~ 2.5 mg/prep

~ 100 µg/prep

Sample volume from overnight culture

2-5 mL

10-150 mL

2-5 mL

Preparation time

5 min

15 min

Concentration of eluted protein

~ 0.3-1 mg/mL

Up to 4.5 mg/mL

~ 0.3-1 mg/mL

Number of purifications

20 reactions

6 preps

96 preps

Use the same buffers for all purification scales – seamless transition

Capturem Miniprep



Capturem Maxiprep



Talon/His60

1.3.2 FLAG-Tag Purification products • Ideal for identifying and monitoring expression of DYKDDDDK- or FLAG-tagged proteins • Highly specific antibody does not cross-react with endogenous proteins • Use beads to purify or immunoprecipitate FLAG-tagged fusion proteins from cell lysates

8



Detection

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY Cat.no.

Description

Size

635686

Anti- DYKDDDDK Beads

1 mL

635687

Immunoprecipitation Buffer Set

39 rxns

635694

Magnetic DYKDDDDK Immunoprecipitation Kit

50 rxns

635695

Anti-DYKDDDDK Magnetic Beads

Each

635696

Magnetic Beads Immunoprecipitation Buffer Set

50 rxns

1.3.3 GST-Tag Purification products • • • • •

Easy to use: No optimization required Purifies more protein: High binding capacity (>10 mg of recombinant GST fusion protein per mL resin) Yields highly purified protein: Eluted protein is free of cellular contaminants Reusable: Easy protocol for regenerating and storing resin Provided in flexible formats: • Bulk resin suitable for batch/gravity-flow purification and FPLC applications • Prepacked gravity-flow columns • Ready-to-use purification kit (5 columns and sufficient reagents for 5 purifications)

Cat.no.

Description

Size

635607

Glutathione-Superflow Resin

10 mL

635608

Glutathione-Superflow Resin

100 mL

635619

GST Purification Kit

5 rxns

1.3.4 Myc-Tagged Protein Purification Monoclonal c-Myc Antibody crosslinked to immobilized protein A agarose or magnetic beads. • Provides quick and easy separation of myc-tagged proteins • Yields highly pure denatured proteins • Highly specific antibody to myc, does not cross-react with endogenous proteins

Cat.no.

Description

Size

631208

c-Myc Monoclonal Antibody-Agarose Beads

1 mL

635698

Magnetic Myc Immunoprecipitation Kit

50 rxns

9

Sample Preparation



Protein Separation





Blotting

AB Incubation



Detection

YY 1.3.5 Non-Tagged protein purification Although protein tagging enables simple, effective affinity purification of a wide variety of proteins, there are a number of reasons why it is not necessarily the best method for every protein: • Tags cannot be used to purify endogenous proteins; they can only be used to purify proteins that have been genetically manipulated, which may not be possible or desirable. • In some cases, a tag may interfere with the structure or biological activity of a target protein, or may be difficult to remove without altering its structure or function. • Protein tagging may be unnecessary for proteins with endogenous post-translational modifications or affinity binding sites, such as phosphoproteins, glycoproteins, or antibodies, which can bind specifically to alternative affinity resins. Phosphoprotein Enrichment Phosphoprotein enrichment with a unique IMAC (immobilized metal affinity chromatography) resin provides an effective affinitybased procedure for isolating phosphorylated proteins from mammalian cells and tissues, using gravity columns or magnetic beads. A similar IMAC resin-based method is available for isolating phosphopeptides, using spin columns or magnetic beads.

Add Extraction/ Loading Buffer (Buffer A) to cell pellet

Total cellular protein

Phosphoprotein Affinity Column

Resin Flowthrough Mix, incubate, and centrifuge

Cell pellet

Load sample

Was and elute with Buffer B

Phosporylated proteins

Cat.no.

Description

Size

635643

Magnetic Phosphopeptide Enrichment Kit

300 rxns

635635

Phosphopeptide Enrichment Buffer Kit

Each

635634

Phosphopeptide Enrichment Spin Columns

Each

635624

Phosphoprotein Enrichment Kit

6 preps

635641

TALON PMAC Magnetic Phospho Enrichment Kit

Each



10

®

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY Glycoprotein Enrichment Glycoprotein enrichment with a boronic acid-based resin provides quick, efficient, and specific enrichment of glycoproteins from complex samples such as human serum, urine, and spinal fluid, using either simple gravity flow columns or medium pressure methods such as FPLC. Enriched glycoproteins transferred to membranes for Western blotting are selectively detected using Glycoprotein Western Detection Kit. It provides highly sensitive and rapid detection—yielding results within one hour.

Cat.no.

Description

Size

635647

Glycoprotein Enrichment Resin

10 mL

635648

Glycoprotein Western Detection Kit

20 rxns

Antibody Purification Antibody purification using thiophilic resins is a simple, powerful, and economical alternative to Protein A, the most common method of immunoglobulin purification. Although Protein A antibody purification is very common, there are certain types of antibodies, such as the single-chain antibodies IgE, IgY, and IgM, that cannot be purified using Protein A. An alternative antibody purification method, thiophilic affinity chromatography (TAC), is ideal for these types of applications, as well as immunoglobulin purification in general, including IgG, IgM, IgA, Fab and Fc fragments, and C3 and C4 complement factors.

Thiophilic Resin Add salt

Filter/ Centrifuge

Load

Wash

Elute in neutral buffer (pH 7.0) 2 days

Protein A Filter/ Centrifuge

Load

Wash

Cat.no

Description

Size

635616

Thiophilic-Superflow Resin

10 mL

635617

Thiophilic-Superflow Resin

100 mL

635614

Thiophilic-Uniflow Resin

100 mL

Elute in low pH buffer (pH ≤ 3.0)

Perform dialysis

Concentrate protein

11

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 1.4 Concentration Measuring After extracting the proteins from the samples and prior to SDS-PAGE, it is important to determine the total protein concentration in each sample. By measuring the concentration, it is possible to ensure the correct amount of protein to be loaded on the gel, thereby minimizing high background and nonspecific antibody binding. Concentration measurements are also useful for semi-quantitative Western blotting – simply load the same amount of protein in each lane and compare the relative levels of the target protein. DeNovix is a new and innovative instrument for measuring concentrations in an easy and intuitive way. Rapidly measure protein samples before proceeding to Western blot analysis. A 0.5 – 1.0 µL sample is all that is required DeNovix advantages for protein studies: • • • • • • •

Fluorometer and spectrophotometer in one instrument! Protein (A280 and labeled) and peptide (A205/215) analysis High absorbance capabilities: up 1125 mg/mL BSA Compatible with Qubit and Quant-it fluorescence assays Built-in cuvette heater controls temperatures for kinetic studies Small footprint: 20 cm x 33 cm Weight: 2 kg

Also ideal for quantifying DNA and RNA samples!

Fluorometer: 0.5 mL thin-walled PCR tube

Cuvette: Standard quartz and disposable cuvettes, full spectrum UV-Vis

Microvolume: 0.5 – 1.0 µL, full spectrum UV-Vis

Cat. no. DS-11 FX +



DS-11 FX



DS-11 +



DS-11



QFX FLUOROMETER

12



√ √

√ √

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 2 Protein Separation After sample preparation, polyacrylamide gel electrophoresis (PAGE) is carried out. Most often, the samples are treated with the negatively charged SDS to unfold and separate the protein content by size. It is also possible to perform non-reducing gel electrophoresis to maintain the native state of the protein. Furthermore, it is helpful to consider the polyacrylamide gel percentage, which is dependent on protein size – larger proteins needs lower acrylamide concentration and vice versa.

2.1 Protein Concentration and Buffer Exchange Prior to protein separation, it is often necessary to concentrate proteins and remove contents, which interferes with gel electrophoresis. The Afyon SDS-PAGE Sample Preparation Kit removes unwanted buffer contents and concentrates protein samples to save you hours in the lab. Superior advantages: • • • • •

Contaminants (GuHCl, urea, ammonium, sulfate etc.) are efficiently removed It takes less than 10 minutes Normalization of protein concentration – final filtrate contains exactly 5 µg Non-toxic Compatible with SDS-PAGE as well as chemiluminescent and fluorescent Western blotting

Afyon protocol in just 10 minutes:

Extract sample



Add 20 µL Afyon Resin to sample*, vortex, centrifuge, remove supernatant (< 5 min)



Suspend pellet in loading buffer, centrifuge with spin filter (< 5 min)



Filtrate contains exactly 5 µg to load directly on gel



Run gel

*Compatible with dilute samples and samples solubilized in GuHCI, detergents, etc.

Cat. no.

Description

Size

K-02101-010

Afyon SDS-PAGE Sample Preparation Kit

10 reactions

K-02101-025

Afyon SDS-PAGE Sample Preparation Kit

25 reactions

13

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 2.2 Sample Loading Buffers Most often, the prepared samples can be stored at -20°C until use. Prior to gel electrophoresis, the samples are prepared with either a reducing or non-reducing loading buffer, which ensures the right viscosity, color for monitoring the loading and running, denaturation, reduction and pH maintenance.

2.2.1 Reducing and Non-reducing Sample Loading Buffers Avoid wasting time and use pre-mixed loading buffers for SDS-PAGE. Simply add your sample to the loading buffer, boil and load. Samples can remain at room temperature for immediate use, or they can be stored at 4°C or -20°C for later analysis. Choose between reducing and non-reducing loading buffers – both are compatible with staining and Western blotting applications.

Cat. no.

Description

Volume

R-03018-B10

Non-reducing protein sample loading buffer (2x)

1 mL

R-03018-B50

Non-reducing protein sample loading buffer (2x)

5 mL

R-03019-B10

Reducing protein sample loading buffer (2x)

1 mL

R-03019-B50

Reducing protein sample loading buffer (2x)

5 mL

2.2.2 Real-time, In-gel Protein Stain After gel electrophoresis, it is important to visualize your protein bands to ensure that the separation is successful. This is known to be a time-consuming and inconvenient process. Visio in-gel protein stain from Advansta helps you to quickly and easily detect protein bands while running your gel. • • • •

Instant results: follow the visible bands while running the gel Sensitive: detect down to 10 ng per band Fast: heat your samples with Visio for 10 minutes and go Compatible: use with both reducing and non-reducing gels. Analyze your results with downstream applications*

Cat. no.

Description

Volume

K-11053-300

Visio Real-Time Protein Stain, 100 lanes or 10 gels

0.3 mL

K-11053-B30

Visio Real-Time Protein Stain, 1,000 lanes or 100 gels

3 mL

*Compatible with mass spectrometry. The product has not been validated with downstream Western blotting, but many customers have had success with it.

14

1) Mix Visio with protein sample

2) Incubation at 98 °C for 10 min, vortex and centrifuge briefly – add reducing buffer for reducing gels

3) Load

4) Run

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 2.3 Gel Electrophoresis Buffers To ensure the right pH, electrical conductivity and a negative charge on the proteins, the right composition of running buffer should be used. Save time with Avant Buffer Pouches. By adding just 500 mL of deionized water and mixing, you will have a freshly made, ultrapure buffer for your SDS-PAGE, which ensures reproducible results.

Cat. no.

Description

Size

R-01038-020

PBS (concentration 1x at 500 mL)

20 pouches

R-01039-020

TBS (concentration 1x at 500 mL)

20 pouches

R-01037-020

Tris-Glycine PAGE running buffer (concentration 1x at 500 mL)

20 pouches

R-01036-020

Tris-Glycine SDS-PAGE running buffer (concentration 1x at 500 mL)

20 pouches

2.4 Molecular Weight Standards In addition to the Western blot signal, knowledge of the protein size is very useful, which can be confirmed with molecular weight standards. From Santa Cruz, we offer a number of standards, which are provided in SDS-PAGE loading buffer for direct use on your gel: • •

Available in low, broad and high range. Use Cruz Marker™ Molecular Weight Standards for visualization on the final Western blot after incubation with the Cruz Marker™-compatible secondary antibodies for Western blotting. Choose between either HRP or AP conjugated secondary antibodies.

Cat. no.

Description

Size

Sc-2035

Cruz Marker™ Molecular Weight Standards

sufficient for 50 gels

Sc-2361

Broad Range Markers

500 µL

Sc-2362

High Range Markers

500 µL

Sc-2360

Low Range Markers

500 µL

15

Sample Preparation





Protein Separation

Blotting



AB Incubation



Detection

YY 2.5 Gel Cast and Run Electrophoresis performance is dependent on a uniformly cast gel and a high-quality running apparatus. UltraCruz® Electrophoresis Cell – simple, safe and easy • • • •

Two-in-one cell suitable for both gel casting and run. Can run up to 10 or 15 samples. Power turns off automatically when lid is opened High resolution separation

Cat. no

Description

Sc-201625

UltraCruz® Electrophoresis Cell

2.6 Protein Stain Check the quality of protein resolution on your gel or the protein transfer on your membrane by staining. Visualize total protein on your gel or blot with the non-toxic fluorescent AdvanStain Scarlet to detect down to 1 ng on laser or CCD imaging systems. Destain your gel and you are ready for downstream Western blotting. Excitation wavelengths include blue, green, violet and UV, while maximum emission wavelength is 610 nm. Fast protocol:

Fix (1 hour)



Stain (1 hour)



Wash (30 min)



Acidify (30 min)

1D and 2D gels stained with excellent sensitivity, large dynamic range, low background and no speckling!

16

Cat. no.

Description

Volume

K-11072-B50

AdvanStain Scarlet Kit, sufficient for 20 minigels

5 mL

K-11072-C25

AdvanStain Scarlet Kit, sufficient for 100 minigels

25 mL



Image

Sample Preparation



Protein Separation





Blotting

AB Incubation



Detection

YY 3 Blotting Blotting is carried out to transfer proteins from the gel to a solid membrane. The blotting is performed by assembling filter papers, a membrane and a gel into a sandwich. An electrical field allows the proteins from the gel to wander onto the membrane.

3.1 Membranes Choose between nitrocellulose and PVDF membranes. We recommend pre-cut membranes for ease of use. Alternatively, cut your membranes from a roll for an economical solution. Use WesternBright transfer membranes for high performance and low background levels. WesternBright membrane characteristics: WesternBright PVDF-FL

WesternBright PVDF-CL

WesternBright NC

Pore size

0.22 µm

0.22 µm

0.22 µm, 0.45 µm

Thickness

135 µm

140 µm

150 µm

Binding interaction

Hydrophobic

Hydrophobic

Hydrophobic, electrostatic

Typical protein binding capacity

300 µg/cm2

300 µg/cm2

200 µg/cm2

Fluorescence Total protein stain

Total protein stain Chromogenic Chemiluminescence

Chemiluminescence Direct staining Radiolabeling Colorimetric

Electrophoretic transfer of proteins

Electrophoretic transfer of proteins

Electrophoretic transfer of proteins Protein dot and slot blotting

Western transfer with fluorescent detection

Western transfer with chemiluminescent detection

Western transfer with chemiluminescent detection Colony/plaque lifts

Pre-cut membranes Membrane rolls

Pre-cut membranes Membrane rolls

Pre-cut membranes Membrane rolls

Detection methods Immobilization methods Recommended applications Available as

Cat. no.

Description

Size

L-08003-010

Pre-cut WesternBright NC 0.22 um, 8 x 10 cm

10 sheets

L-08002-010

Pre-cut WesternBright NC 0.45 um, 8 x 10 cm

10 sheets

L-08004-010

Pre-cut WesternBright PVDF-CL, 7 x 9 cm

10 sheets

L-08001-010

Pre-cut WesternBright PVDF-FL, 7 x 9 cm

10 sheets

L-08012-010

Pre-cut WesternBright PVDF-FL, 10 x 15 cm

10 sheets

L-08011-010

Pre-cut WesternBright PVDF-CL, 10 x 15 cm

10 sheets

L-08014-010

Pre-cut WesternBright PVDF-FL, 13 x 18 cm

10 sheets

L-08013-010

Pre-cut WesternBright PVDF-CL, 13 x 18 cm

10 sheets

L-08006-001

WesternBright NC membrane roll, 0.22 μm, 30 cm x 3 m

1 roll

L-08007-001

WesternBright PVDF-FL membrane roll, 0.22 μm, 26 cm x 3.3 m

1 roll

L-08008-001

WesternBright PVDF-CL membrane roll, 0.22 μm, 26 cm x 3.3 m

1 roll

17

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 3.2 Blotting Papers Standardized, pre-cut blotting papers save you time during sandwich assembly. We offer blotting papers with useful advantages: • •

Pre-cut papers available in two sizes Uniform thickness ensures reproducible transfers without introducing artifacts or background noise

Cat. no.

Description

Size

L-07045-060

Blotting papers, 7 x 9 cm

60 sheets

L-07056-060

Blotting papers, 8 x 10 cm

60 sheets

3.3 Transfer Buffer It is important to choose a high-quality transfer buffer to optimize the detection of low-abundance proteins, the transfer of high-molecular weight proteins and to save time. Optimize the transfer from gel to membrane with Advansta’s proprietary FLASHBlot Transfer Buffer: • •

Ideal for low-abundance protein samples and post-translationally modified proteins Transfer proteins of all sizes in less than 20 minutes

CEA (high MW) and GAPDH (low MW) Western blots. The proteins were transferred for 15 minutes using a FLASHBlot buffer and 60 minutes with a Towbin buffer, respectively. Transfers for both CEA and GAPDH were most efficient when using FLASHBlot.

18

Cat. no.

Description

Size

R-03090-D25

FLASHBlot Transfer Buffer, 50x

250 mL

R-03090-D50

FLASHBlot Transfer Buffer, 50x

500 mL

Sample Preparation





Protein Separation

Blotting



AB Incubation



Detection

YY 3.4 Protein Stain for Membranes Before proceeding with antibody incubation, it is advantageous to check the quality of the protein transfer. Advantages with AdvanStain Ponceau: • • •

Stains your membrane in less than 5 min. Destain the membrane just as easily Following destaining, the membrane is ready for incubation with antibodies

Cat. no.

Description

Size

R-03021-D50

AdvanStain Ponceau

500 mL

19

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 4 Incubation with Antibodies Once the proteins have been transferred to a membrane, it needs to be blocked, incubated with a primary antibody, washed, incubated with a secondary antibody and then washed a final time. Instead of using both primary and secondary antibodies, direct detection can, in many cases, be more suitable.

4.1 Blocking Solution Avoid non-specific binding of antibodies by using an appropriate blocking solution. AdvanBlock-PF offers: • • • •

Non-protein blocking solution No cross-reactivity with primary antibodies as seen with nonfat dry milk, BSA or casein Compatible with both fluorescent and chemiluminescent detection Optimized for use with WesternBright fluorescent secondary antibodies

Cat. no.

Description

Size

R-03023-D20

AdvanBlock-PF Blocking Solution, 5x

200 mL

4.2 Washing Buffer for Western Blots After each incubation step with antibodies, the blot needs to be washed with an appropriate washing buffer. Use AdvanWash washing solution whether you are performing fluorescent or chemiluminescent Western blotting. The solutions can be used with any Western blotting systems, but are optimized for use with WesternBright chemiluminescent HRP substrates, WesternBright MCF fluorescent Western blotting kit and WesternBright MCF-IR kits. • Reliable: Quality-controlled for reproducible results • Versatile: Compatible with chemiluminescent and fluorescent Western blots • Convenient: Save time using pre-made buffers

Cat. no.

Description

Size

R-03024-D50

AdvanWash Washing Solution, 10x

500 mL

R-03100-D50

AdvanWash-IR Washing Solution, 10x

500 mL

4.3 Direct or Indirect Methods? Direct detection relies on a single primary antibody containing a detectable label, while indirect detection requires both a primary antibody as well as a secondary antibody. In indirect targeting, the secondary antibody contains the label.

20

Sample Preparation



Protein Separation





Blotting

AB Incubation



Detection

YY Indirect

Direct

Advantages

• Signal amplification – multiple secondary • Fewer steps antibodies bind to primary antibody • Less potential for non-specific binding • Versatile – same secondary antibody can be used on different primary antibody of same species

Disadvantages

• Risk of non-specific binding • More steps

• Can be more costly • Labeling of antibody can affect the immunoreactivity

4.4 Primary Antibodies When performing Western blots, it is important to choose antibodies that recognize only the epitopes of interest. Follow the table below for advice on whether to choose monoclonal or polyclonal antibodies: Monoclonal antibodies

Polyclonal antibodies

Recognition of epitope • Recognize a single epitope

• Recognize multiple epitopes within same antigen

Sensitivity

• Less sensitive

• More sensitive – good for low abundance antigens

Cross-reactivity

• Less likely • Potential for recognizing other antigens with the epitope

• More likely • Higher background than monoclonal antibodies • Can cross-react with multiple animal species

Variability

• Very stable batches from same hybridoma

• Variability-prone between batches

Tolerance*

• Less tolerant

• More tolerant

*Tolerance against differing conditions, such as denaturing conditions, modifications or presence of polymorphism

4.5 Secondary Antibodies For indirect detection, the blot is treated with secondary antibodies after primary antibody incubation. The primary antibody species determines the choice of secondary antibodies. For chemiluminescence, secondary antibodies are labeled with HRP, while for fluorescence they are labeled with a fluorophore. We offer secondary antibodies from a variety of species with different conjugates. HRP-conjugated secondary antibodies for chemiluminescent detection:

Cat. no.

Description

Size

R-05071-500

Goat anti-mouse HRP-conjugated secondary antibody

500 µL

R-05072-500

Goat anti-rabbit HRP-conjugated secondary antibody

500 µL

R-05073-500

Goat anti-human HRP-conjugated secondary antibody

500 µL

R-05074-500

Goat anti-chicken HRP-conjugated secondary antibody

500 µL

R-05075-500

Goat anti-rat HRP-conjugated secondary antibody

500 µL

R-05076-500

Goat anti-guinea pig HRP-conjugated secondary antibody

500 µL

21

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY Fluorescent secondary antibodies, excitable with visible and near infrared light:

Cat. no

Description, visible light

Size

R-05051-050

Goat anti-rabbit IgG APC

50 µL

R-05051-250

Goat anti-rabbit IgG APC

250 µL

R-05052-050

Goat anti-mouse IgG RPE

50 µL

R-05052-250

Goat anti-mouse IgG RPE

250 µL

Cat. no.

Description, infrared light

Size

R-05054-250

Goat anti-rabbit IgG IR700

250 µL

R-05055-250

Goat anti-mouse IgG IR700

250 µL

R-05056-250

Goat anti-human IgG IR700

250 µL

R-05057-250

Goat anti-chicken IgY IR700

250 µL

R-05058-250

Goat anti-rat IgG IR700

250 µL

R-05059-250

Goat anti-guinea pig IgG IR700

250 µL

R-05060-250

Goat anti-rabbit IgG IR800

250 µL

R-05061-250

Goat anti-mouse IgG IR800

250 µL

R-05062-250

Goat anti-human IgG IR800

250 µL

R-05063-250

Goat anti-chicken IgY IR800

250 µL

R-05064-250

Goat anti-rat IgG IR800

250 µL

R-05065-250

Goat anti-guinea pig IgG IR800

250 µL

Secondary antibodies for a large selection of species: From Santa Cruz Biotechnology we offer a wide variety of conventional secondary antibodies for a large selection of species, raised in either rabbits, goats, donkeys, bovines, mice or chickens.

• Anti-Goat • Anti-Mouse • Anti-Rabbit

• Anti-Rat • Anti-Bovine • Anti-Cat

• Anti-Chicken • Anti-Dog • Anti-Guinea Pig

• Anti-Armenian Hamster • Anti-Horse • Anti-Human

• Anti-Monkey • Anti-Swine • Anti-Turkey

• Anti-Sheep • Anti-Syrian Hamster • Anti-Donkey

Available conjugated to either an enzyme, biotin or fluorophore.

Cruz Marker™-compatible Western blotting secondary antibodies and Cruz Marker™ Molecular Weight Standards. Cruz Markers™ are provided for use as internal molecular weight standards for chemiluminescence Western blotting applications, and they are ideal for approximating the molecular weight of target proteins while allowing researchers to visualize the final molecular weight ladder of six bands clearly and consistently on the final Western blot film. Cruz Marker™ compatible secondary antibodies recognize an epitope common to each of the Cruz Marker™ molecular weights. Pre-adsorbed secondary antibodies are recommended for Western blotting of immunoglobulin-rich tissues and cells.

22

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY

Cruz Marker™ Molecular Weight Standards* Description

Cat. no

Size

Cruz Marker™ Molecular Weight Standards

sc-2035

200 µL

*Must be used in connection with Cruz Marker™-compatible Western blotting secondary antibodies.

Cruz Marker™-compatible Western blotting secondary antibodies* Description

HRP conjugate

AP conjugate

HRP conjugate Pre-adsorbed

AP conjugate Pre-adsorbed

Bovine anti-goat IgG

sc-2378

sc-2381

sc-2384

sc-2387

Donkey anti-goat IgG

sc-2033

sc-2037

sc-2304

sc-2310

Bovine anti-mouse IgG

sc-2380

sc-2383

sc-2386

sc-2389

Donkey anti-mouse IgG

sc-2318

sc-2320

sc-2306

sc-2312

Goat anti-mouse IgG

sc-2031

sc-2047

sc-2302

sc-2308

Bovine anti-rabbit IgG

sc-2379

sc-2382

sc-2385

sc-2388

Donkey anti-rabbit IgG

sc-2317

sc-2319

sc-2305

sc-2311

Goat anti-rabbit IgG

sc-2030

sc-2034

sc-2301

sc-2307

Goat anti-rat IgG

sc-2032

sc-2036

sc-2303

sc-2309

Rabbit anti-mouse IgG

sc-358917

sc-358923

*Are supplied at 200 µg in 0.5 mL, to be used in Western blotting at a dilution of 1:500–1:5000.

4.6 Streptavidin Conjugates The strong interaction between streptavidin and biotin can be utilized for Western blotting. Using streptavidin conjugates enables fluorescent detection of biotinylated proteins. Fluorescent streptavidin conjugates:

Cat. no.

Description

Size

R-05011-050

AdvanFluor APC-Streptavidin conjugate

50 µL

R-05012-050

AdvanFluor BPE-Streptavidin conjugate

50 µL

R-05011-200

AdvanFluor APC-Streptavidin conjugate

200 µL

R-05012-200

AdvanFluor BPE-Streptavidin conjugate

200 µL

We offer over 70,000 monoclonal and polyclonal primary and secondary Western blot antibodies from our suppliers. Antibodies for cancer research, cardiovascular research, drug discovery, immunology, metabolism, neuroscience research, etc.

23

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 5 Detection The detection method is dependent on the label of the antibodies. Using a fluorescence dye enables detectable excitation by light. For chemiluminescent detection, HRP-labeled antibodies are treated with substrate, which produces the detectable signal.

5.1 HRP Substrates Most Western blot users have special needs for HRP substrates, including high sensitivity, long-lasting signal, fast detection and higher dynamic range. Choose the right product for the chemiluminescent detection of your target proteins.

24

Substrate

Use

Sensitivity

Signal duration

WesternBright ECL

Sensitive, economical choice for routine Western blotting

Low picograms

6-8 hours

WesternBright Quantum

Extended dynamic range for detecting low and high abundance proteins within the same blots

Mid femtograms

10-24 hours

WesternBright Sirius

Low abundance protein detection

Low femtograms

6 hours

Cat. no.

Description

Size

K-12045-D20

WesternBright ECL

200 mL

K-12045-D50

WesternBright ECL

500 mL

K-12049-D50

WesternBright ECL spray

500 mL

K-12042-D10

WesternBright Quantum

100 mL

K-12042-D20

WesternBright Quantum

200 mL

K-12043-D10

WesternBright Sirius

100 mL

K-12043-D20

WesternBright Sirius

200 mL

Sample Preparation



Protein Separation



Blotting



AB Incubation



Detection

YY 5.2 How to Choose the Right Chemistry for Your Western Blot The best chemistry for your Western blot depends on the type of information you are looking for.

5.2.1 Chemiluminescence Chemiluminescent Westerns are relatively easy to do and can be extremely sensitive; substrates are available to detect proteins in the femtogram range. “Chemi” is a convenient chemistry if proteins of interest differ significantly in molecular weight and are easily resolved by gel electrophoresis. Advantages:

Disadvantages:

• Sensitive • Easy, familiar chemistry • Compatible with film or digital imaging

• Semi-Quantitative • Signal dependent on enzyme kinetics • Single protein only, loading controls require stripping and re-probing

Use Chemiluminescence to: • • • • •

Detect a single protein Assay for presence/absence of a protein Measure antibody responses Follow protein purification Detect low abundance proteins Chemiluminescence. One signal. One protein

5.2.2 Fluorescence Fluorescent Western blotting uses secondary antibodies conjugated to fluorescent dyes. The amount of light emitted from excited fluorophores is proportional to the amount of target protein on the membrane. This allows for true quantitative analysis. Using fluorophores with different emission spectra, one can detect multiple proteins on a blot simultaneously (multiplexing). Loading controls can be measured on the same blot as your protein of interest, along with post-translational modifications without stripping and re-probing. Fluorescent Westerns use either Infrared or Visible fluorophores. Advantages:

Disadvantages:

• Multiplex capability • Increased quantitative accuracy • Fluorescent label stability allows blots to be stored and re-imaged later

• Can be less sensitive than chemiluminescence • Membrane auto-fluorescence can increase background

Advantages of Visible Fluorescent Detection If you need to study three proteins at once, visible fluorescence is the obvious choice. Visible fluorescence also offers flexibility. There are many choices of fluorophores available for visible detection. If you are already using (spectrally separated) visible fluorophores for other experiments, adapting these for use in fluorescent Western blots is easy.

25



Sample Preparation

Protein Separation



Blotting



AB Incubation



Detection

YY Use Fluorescence to: • • • • •

Detect multiple proteins simultaneously Study post-translational modifications Have same-blot loading control Have in-lane normalization Perform quantitative Westerns Multiplex Fluorescence. Multiple Proteins

The Azure c Series Imaging Systems The Azure Biosystems cSeries offers 6 unique gel imaging systems. Select the one that fits your applications now, and upgrade later when your needs change. • A total solution for Western Blot Imaging and Gel Documentation: The cSeries is a multichannel, multimodal imager, with IR, visible light, and UV excitation channels. • Compact, integrated design: A unique design and integrated tablet computer gives this system a small footprint, saving you room for other tools on the bench top. • Fully upgradable: All models can be easily upgraded at a later time to accommodate additional applications. • 8.3MP CCD camera for sensitive detection of chemiluminescence: 5 levels of binning and a Chemi Blot Shelf to bring the blot closer to the detector, if needed. • 16 bit camera: c-series has a wide dynamic range, preventing saturation and keeping results in linear range. • Deep Peltier cooling of the camera: Absolute and Regulated cooling improves image quality by reducing the noise in the image. • AzureSpot Analysis Software: Analysis tool for 2D densitometry, automatic lane and band detection, background correction, normalization, molecular weight calculation, and annotation.

c600



c500



































c200







c150







c400 c300

26

√ √

6 Troubleshooting 6.1 Unusual/Unexpected Bands Problem Lower MW than expected Slightly higher MW than expected Significantly higher MW than expected

Cause

Solution

• Digestion/cleavage of target

• Use fresh sample and protease inhibitors

• Splice variant exist or other proteins bear similar epitope

• Consult literature and use appropriate controls • Try another antibody

• Potential for glycosylated protein or small amino acid modifications

• Use enzymes to remove the modification • Check amino acid sequence and literature

• Dimers/multimers/protein-protein interactions occur due to inefficient reduction and denaturation

• Use fresh DTT or β-mercaptoethanol for samples + reheat before repeating experiment • Prepare new samples with fresh loading buffer

• Changed protein expression during over-passaged cell line

• Use earlier passages, include positive control

• Excessively high concentration of primary antibody or occurrence of cross-reactivity with similar epitopes on other proteins

• • • •

• Non-specific binding of secondary antibody

• Optimize secondary antibody concentration • Use affinity-purified secondary antibody • Check non-specific binding by repeating immunodetection with secondary antibody alone

• Voltage too high during gel run

• Run gel at lower voltage

• Slow migration

• Increase voltage, ensure proper buffer preparation

• Incorrect heating of sample

• Ensure heating to 90 °C for 2 min prior to loading

• Old SDS in sample buffer

• Use fresh SDS for sample buffer

• Incorrect buffer composition

• Use fresh buffer

• Bubbles trapped between gel and membrane during transfer

• Carefully remove air bubbles

• Overheated gel due to fast running conditions

• Optimize electrophoresis conditions • Run gel at 4 °C

• Too much protein loaded

• Decrease amount of sample • Repeat with dilution series of sample

• Antibody concentration too high

• Optimize antibody concentrations

• High salt concentration in sample

• Decrease salt concentration in sample buffer

• Sample concentration too high/insufficient SDS

• Increase dilution/add more SDS

• Diffusion of samples from well to well

• Minimize loading time

• Incomplete polymerization of gel around wells

• Increase TEMED/AP

• High pressure applied to gel during pouring

• Loosen screws on the gel assembly apparatus

• Particulate matter in gel

• Filter and mix gel reagents prior to gel preparation

• Excessive/uneven heating of gel

• Decrease running voltage/apply cooling during run

• Bubbles in gel due to dirty plates

• Wear gloves during plate handling • Clean plates thoroughly with ethanol and deionized water

• Bubbles in gel from air introduced from pouring device

• Do not expel entire volume of gel mix

• Bubbles under comb

• Insert comb at an angle and reposition before gel solidification

Multiple bands

Blurry/diffuse bands

Smiling bands (not flat) White bands Streaking in lanes Lateral spreading of bands

Band distortion

Use affinity-purified primary antibody Optimize primary antibody concentration Try another Check antibody specificity with blocking peptide

27

6.2 No Bands, Faint Bands or Weak Signal Problem

Sample preparation

Inadequate transfer

Inefficient binding of primary antibody

Inefficient binding of secondary antibody Inactive conjugate/substrate Detection methods

Cause

Solution

• Inefficient extraction method

• Use other extraction methods • Include positive control

• Low expression levels of protein

• Load more protein, concentrate extracted proteins, pool multiple samples

• Degraded protein during degradation

• Use fresh protease inhibitor

• Wrong transfer buffer composition, too much methanol in buffer

• Check protocol, decrease methanol concentration

• Large protein requires more time and/or current

• Run for longer time and/or with higher voltage

• No contact between gel and membrane

• Check fiber pad thickness, replace if too thin

• Protein has transferred through the membrane

• Use two membranes

• Old/weak antibody, low affinity of antibody for protein

• Increase primary antibody concentration, purchase fresh antibody, reduce number of wash steps

• Weak cross-reactivity with species of interest

• Try other primary antibody source

• Antibody removed with washing steps

• Decrease number of washes, decrease salt concentration in wash buffer, reduce washing time

• Antigen masked by blocking agent

• Use alternative blocker • Try lower concentration of blocker

• Inappropriate secondary antibody

• Use antibody directed against primary antibody species

• Insufficient antibody concentration/antibody too old

• Purchase new antibody • Increase dilution

• Old/unstable reagents

• Check conjugate and substrate for signal • Purchase new reagent

• HRP inactivation by sodium azide

• Avoid sodium azide

• Old detection reagent/improper storage of detecting reagent

• Purchase new reagent

• Exposure time too short

• Test different exposure times

6.3 Electrophoretic Problems Problem

Cause

Solution

• Insufficient TEMEP/AP

• Increase amount of TEMEP/AP

• Old AP solution

• Prepare fresh AP and store only for a few days at 4 °C

• Polymerization inhibition due to oxygen

• Layer gel with isopropanol prior to pouring stacker

• Excessive amount of TEMED/AP

• Decrease amount of TEMED/AP

• Concentration of running buffer too high

• Check protocol/dilute buffer

• Insufficient current

• Increase voltage

• Buffer excessively diluted

• Check protocol/replace buffer

Smiling dye front

• Migration too fast

• Decrease voltage

• Heat generation

• Run with cooling

Slanted dye front

• Bubble between glass plates at the bottom of the gel

• Hold gel at an angle, place corner into lower buffer chamber and slowly move to normal position

Slow/No gel polymerization Too fast gel polymerization Very long run time Running time unusually short

28

6.4 High Background Problem

Membrane blocking is insufficient

Inappropriate wash conditions Reagant contamination

Membrane choice

Overexposed image

Cause

Solution

• Biotin in milk incompatible with streptavidin system/milk contains antigen of interest

• Try another blocking agent

• Milk solution excessively diluted

• Increase milk solution

• Blocking time too short

• Increase incubation time

• Some detergents are not as effective at cold temperatures

• Incubate for 1 hour at room temperature instead of overnight at 4 °C

• Insufficient detergent concentration

• Use stronger detergents/increase detergent concentration

• Insufficient washing

• Increase duration of washing steps, use larger volume of washing buffer, increase number of washing steps • Try other incubation temperatures

• Bacterial/fungal growth in buffers

• Check turbidity in buffers • Prepare new buffers

• PVDF membranes may have higher background than nitrocellulose

• Try nitrocellulose membranes

• High autofluorescence in membrane

• Use low autofluorescence PVDF membranes for fluorescent Western blots

• Membrane has dried out

• Ensure hydration during all steps by using sufficient volumes and agitation

• Exposure time too long

• Reduce exposure time, increase antibody dilutions • Load less sample

Non-specific bin- • Antibody concentration too high/antibody not affinity purified ding of primary or secondary • Too much protein on gel antibodies

• Decrease antibody concentration/try monoclonal or affinity-purified antibody/optimize incubation times • Load less protein

29

Contact Denmark blå tynd streg

hvid alm. streg

blå alm. streg

pink tynd streg

+45 8745 9010 www.ahdiagnostics.dk [email protected]

Sweden blå tynd streg

hvid alm. streg

blå alm. streg

pink tynd streg

+46 08 680 08 45 www.ahdiagnostics.se [email protected]

Norway blå tynd streg

hvid alm. streg

blå alm. streg

pink tynd streg

+47 2323 3260 www.ahdiagnostics.no [email protected]

Finland blå tynd streg

hvid alm. streg

blå alm. streg

pink tynd streg

+358 8 010 325 3000 www.ahdiagnostics.fi [email protected]

30

31

AH diagnostics as • Runetoften 18 • DK-8210 Aarhus V • Tel.: +45 8745 9010 • [email protected] • www.ahdiagnostics.dk AH diagnostics AB • Vallgatan 9 • SE-170 67 Solna • Tel.: +46 (0)8 680 0845 • [email protected] • www.ahdiagnostics.se AH diagnostics Oy • Viikinkaari 4 • FI-00790 Helsinki • Tel.: +358 (0)10 325 3000 • [email protected] • www.ahdiagnostics.fi AH diagnostics as • Fjellgata 1 • NO-0566 Oslo • Tel.: +47 2323 3260 • [email protected] • www.ahdiagnostics.no

D

S

N

F