SDS-PAGE and western blot for high molecular weight proteins (30-500kDa) Merav Marom Shamur, Smart Assays Aim: Analysis of high molecular weight proteins by SDS-PAGE and western blot under reducing conditions. Control: negative control: Positive control: Instrument Product name PowerPac Basic Power supply Xcell SureLock Mini-Cell Mini trans blot cell

Company Bio Rad Life technologies Bio Rad

Cat # 165-6019 EI0001 170-3930

Reagents Product name Nupage 3-8% Tris Acetate gel 20X Nupage Tris Acetate SDS Running buffer InstantBlue (Coomassie) HiMark pre-stained Protein standard Tris pH 6.8 Glycerol SDS Beta mercapto Ethanol Bromo phenol blue Methanol Purified HA-protein X (150 kDa, 1µg/µl) Purified HA-protein Y (250 kDa, 1.5µg/µl) Mouse anti HA AP Goat anti mouse-HRP Skim milk 20X Transfer buffer PBSX10 Tween-20 Ponceau Developer solution Fixation solution SuperSignal X-Ray film Blot Absorbent Filter Paper 0.45µm Nitrocellulose membrane

Company

Cat #

Storage

Invitrogen Invitrogen Expedeon Invitrogen Bio Rad Sigma Bio Rad Sigma Sigma Sigma

EA0375 LA0041 ISB1L LC5699 161-0799 G7893 161-0418 M3148 114391 34860

Sigma Jackson Svenska Labfab Invitrogen Biological Industries Sigma Sigma Fuji Fuji Thermo Fuji Bio Rad Bio Rad

H3663 115-035-062 ACU-7352A NP0006 02-023-5A P9416 P7170 DevRTU FixRTU 34077 F1318 170-3932 162-0113

4°C RT 4°C -20°C RT RT RT RT RT RT -80°C -80°C 4°C -20°C RT RT RT RT RT RT RT 4°C RT RT RT

Buffers preparation Buffer 1.

Preparation

Sample buffer 6X (SBX6) Running buffer: 1X Tris/Acetate/SDS buffer Transfer buffer 1X PBS 1X Washing buffer: (PBST) Blocking buffer (5% milk in PBST) Antibody dilution buffer

2. 3. 4. 5. 6. 7.

Storage

140µl 500mM Tris pH 6.8, 300µl 100% glycerol, 240µl 10% SDS, 60µl 100% beta-mercaptoethanol, bromophenol blue, 260µl DDW.

-20µL

50 ml of 20X Tris/Acetate/SDS into 950 ml of DDW

RT

50 ml of 20X Transfer buffer, 200ml Methanol, 750 ml DDW. 100ml of PBSX10 into 900 ml DDW Add 1.25 ml 20% Tween-20 into 500 ml buffer prepared in 4

RT RT RT

Weight 2.5 gr of skim milk and add 50ml of PBST

RT

Weight 1.25gr of skim milk and add 25ml PBS

RT

Procedure: General gel map: 1

2

SBX1

SBX1

SDS-PAGE 3 4 1µg 1µg Protein Protein X Y

5

6

Marker

SBX1

Western blot 7 8 100ng 100ng Protein Protein X Y

9

10

Marker

SBX1

Samples preparation: 1. Prepare sample buffer X6 as described in buffer preparation section (1) 2. Prepare samples as follows (volume taken marked in red): 2.1 First dilute protein X and protein Y 1:10 (take 1µl of protein and add 9µl of DDW) to get protein X 0.1µg/µl, protein Y 0.15µg/µl.

Protein stock Protein (µl) SBX6 (µl) DDW (µl)

Protein X 1µg/µl 0.1µg/µl 1µg 100ng 1 1 4 4 19 19

Protein Y (1.5µg/µl) 1.5µg/µl 0.15µg/µl 1µg 100ng 0.7 0.7 4 4 19.3 19.3

3. Incubate samples at 95°C for 5 min.

Assembling the Gel System: 1. Spread white soft paraffin around both of the silicone gaskets of the buffer cure to avoid buffer leakage.

2. Lower the buffer core into the lower buffer chamber so that the negative electrode fits into the opening in the gold plate on the lower buffer chamber 3. Insert the Gel Tension Wedge into the XCell SureLock™ behind the buffer core. Make sure the Gel Tension Wedge is in its unlocked position, allowing the wedge to slip easily into the XCell SureLock™ unit (figure 1).

4. Insert gel cassettes into the lower buffer chamber. 5. Place one cassette (or buffer dam) behind the core and one cassette in front of the core. For each cassette, the shorter “well” side of the cassette faces in towards the buffer core. The slot on the back of the cassette must face out towards the lower buffer chamber 6. Pull forward on towards the front of the XCell SureLock™ unit lever comes to a firm stop and the gels or gel/buffer dam appear snug against the buffer core (figure 1).

Figure 1: Assembling the XCell SureLock Mini cell

7. Prepare running buffer as described in buffer preparation section (2). 8. Fill the upper buffer chamber with ~200 ml running buffer. Use enough running buffer to completely cover the sample wells. 9. Ensure that the Upper Buffer Chamber is not leaking. 10. Fill the lower buffer chamber with ~600 ml running buffer.

Loading samples 11. Use gel loading tips to underlay samples and marker into the gel wells 12. Lower the tip to the bottom of the well and slowly pipette sample into well as mentioned above in general gel map.

Running Conditions 1. 2. 3.

Place the lid on the chamber and plug the lid into the power source. Apply a constant voltage of 150 V for 60 min. The expected start current is 40–55 mA/gel and the expected end current is 25–40 mA/gel.

Disassembling the gel system 1. At the end of the run, turn off the power and disconnect the cables from the power supply. 2. Remove the lid and unlock the Gel Tension Lever. 3. Remove the gel cassette from the gel system

4. Lay the gel cassette on a flat surface 5. Carefully insert the Gel Knife's beveled edge into the narrow gap between the two gaps of the cassette 6. Push up and down gently on the knife's handle to separate the plates (cracking sound will be heard) 7. Rotate the cassette and repeat separating the plates until the two plates are completely separated. 8. Upon opening the cassette, the gel may adhere to one of the two plates of the cassette 9. Gently remove gel wells and foot by Gel Knife: hold the Gel Knife at a 90° angel to the gel and. Push straight on the knife to cut the gel. 10. Cut the gel into two parts : one for SDS-PAGE staining and second for western blot as follows (dashed red line): SDS-PAGE Western blot 1 2 3 4 5 6 7 8 9 10 1µg 1µg 100ng 100ng SBX1 SBX1 Protein Protein Marker SBX1 Protein Protein Marker SBX1 X Y X Y

Staining procedure 1. Rinse the SDS-PAGE gel in InstantBlue solution. 2. Using a pipette and gently peel the gel away from the plate into a plastic box containing 20ml Instantblue solution. 3. Incubate for 15 min at RT with gentle shaking. 4. Scan gel. Transfer and Blotting 1. Prepare transfer buffer as described in buffer preparation section (3). 2. Equilibrate nitrocellulose membrane and two fiber pads in transfer buffer for 5-10 min. 3. Gently, remove the western blot gel from the plate using a tip or clean spatula around the gel. 4. Place the cassette, with the black side down, on a clean surface. 5. Place one pre-wetted fiber pad on the black side of the cassette. 6. Place a two sheets of filter paper on the fiber pad. 7. Place the equilibrated gel on the filter paper. 8. Place the equilibrated membrane on the gel. 9. Complete the sandwich by placing a 2 pieces of filter paper on the membrane. 10. Add the last fiber pad. 11. Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the white latch. 12. Place the cassette in module. Repeat for the other cassette. 13. Add 1 liter of chilled transfer buffer to the tank. 14. Insert pre-freezed Bio-Ice cooling unit to the tank. 15. Place the lid on top of the buffer tank cell. 16. Connect the cables to the power supply 17. Apply a constant current of 45mA for overnight at RT.

Ponceau staining 1. Prepare PBSX1 as described in buffer preparation section (4) 2. Prepare PBST as described in buffer preparation section (5). 3. Carefully remove the membrane from transfer sandwich into DDW containing box. 4. Wash with gentle agitation for 5 min 5. Remove DDW 6. Add ponceau solution and shake gently until the marker bands and sample protein are visualized. 7. Add DDW to remove nonspecific staining 8. Remove DDW 9. Carefully place the membrane between two clean transparent plastic sheets 10. scan membrane 11. Carefully remove the membrane into a clean box containing PBST 12. Wash with gentle agitation until all ponceau solution is removed from the membrane (washing solution may be replace few times if needed). Blocking 1. Once all ponceau solution is removed from membrane, discard washing solution. 2. Prepare blocking solution as described in buffer preparation (6). 3. Apply blocking solution ~10ml. 4. Incubate 1 hour at RT on a platform rotator. 1st Ab 1. Discard blocking solution 2. Add 10 ml of 1:1000 diluted anti HA antibody in antibody dilution buffer (7). 3. Incubate the membrane overnight at 4°C on rotating platform

Washings 1. Discard 1st Ab solution 2. Add 10ml of washing buffer 3. Incubate for 5 minutes, on a platform rotator. 4. Discard washing solution 5. Repeat steps 1-4 two more times 2nd Ab 1. Discard washing solution 2. Add 10ml 1:10,000 diluted Peroxidase conjugated Goat anti mouse in blocking buffer. 3. Incubate 1h at RT on rotating platform Washings 4. Discard 1st Ab solution 5. Add 10ml of washing buffer 6. Incubate for 5 minutes, on a platform rotator.

7. Discard washing solution 8. Repeat steps 1-4 two more times

Detection *Note: Perform this step inside a dark room with red light 1. Mix the two SuperSignal substrate components at a 1:1 ratio to prepare the substrate Working Solution: use 1ml of each solution (total volume:2ml) 2. Discard washing solution 3. Apply (1:1) diluted SuperSignal Substrate on the membrane. 4. Incubate 5 min at RT 5. Drain excess reagent. 6. Carefully hold the membrane using a tweezers 7. Place the membrane between two transparent plastic sheets inside the x-ray cassette. 8. Expose blot to X-ray film by placing the x-ray film on top of the transparent plastic sheet containing the membrane Developing 1. Remove x-ray film from cassette and place film inside a developer containing glass vessel 2. Agitate gently until black bands are visualized. 3. Remove film from developer solution 4. Shake film gently to remove excesses of developer solution 5. Wash film using water containing vessel 6. Shake film gently to remove excesses of water 7. Place film in fixing solution containing glass vessel 8. Agitate gently for 1 minute 9. Remove film from fixing solution 10. Shake film gently to remove excesses of fixing solution 11. Place film in water containing vessel 12. Air dry film 13. Mark the specific position of MW protein marker 14. Scan film