Product name Company Cat # PowerPac Basic Power supply Bio Rad Mini Protean electrophoresis system Mini trans blot cell Bio Rad

Transcription

1 SDS-PAGE and western blot for low molecular weight proteins (2-20kDa) Merav Marom Shamur, Smart Assays Aim: Analysis of low molecular weight proteins by SDS-PAGE and western blot under reducing conditions. Control: negative control: Positive control: Instrument Product name Company Cat # PowerPac Basic Power supply Bio Rad Mini Protean electrophoresis system Bio Rad Mini trans blot cell Bio Rad Reagents Product name Company Cat # Storage 16.5% Tris Tricine gel Bio Rad C 10X Tris/Tricine/SDS buffer Bio Rad RT InstantBlue (Coomassie) Expedeon ISB1L 4 C marker Bio Rad C Tris ph 6.8 Bio Rad RT Glycerol Sigma G7893 RT SDS Bio Rad RT Beta mercapto Ethanol Sigma M3148 RT Bromo phenol blue Sigma RT Methanol Sigma RT Purified V5-protein X (10 kda, 1µg/µl) -80 C Purified V5 -protein Y (15 kda, 1.5µg/µl) -80 C Mouse anti V5 Invitrogen R960 4 C Goat anti mouse-hrp Jackson C Skim milk Svenska Labfab ACU-7352A RT 10X Tris / Glycine Bio Rad RT PBSX10 Biological Industries A RT Tween-20 Sigma P9416 RT Ponceau Sigma P7170 RT Developer solution Fuji DevRTU RT Fixation solution Fuji FixRTU RT SuperSignal Thermo C X-Ray film Fuji F1318 RT Blot Absorbent Filter Paper Bio Rad RT Immobilon-P membrane, PVDF milipore IPVF00010 RT

2 Buffers preparation Buffer Preparation Storage 1. Sample buffer 6X (SBX6) 140µl 500mM Tris ph 6.8, 300µl 100% glycerol, 240µl 10% SDS, 60µl 100% beta-mercaptoethanol, bromophenol blue, -20µL 260µl DDW. 2. Running buffer: 1X Tris/Tricine/SDS buffer 100 ml of 10X Tris/Tricine/SDS into 900 ml of DDW RT 3. Transfer buffer 1X 100 ml of 10X Tris / Glycine, 200ml Methanol, 700 ml DDW. RT 4. PBS 1X 100ml of PBSX10 into 900 ml DDW RT 5. Washing buffer: (PBST) Add 1.25 ml 20% Tween-20 into 500 ml buffer prepared in 4 RT 6. Blocking buffer (5% milk in PBST) Weight 2.5 gr of skim milk and add 50ml of PBST RT Procedure: General gel map: SDS-PAGE Western blot SBX1 SBX1 1µg X 1µg Y Marker SBX1 100ng X 100ng Y 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. X Y (1.5µg/µl) stock 1µg/µl 0.1µg/µl 1.5µg/µl 0.15µg/µl 1µg 100ng 1µg 100ng (µl) SBX6 (µl) DDW (µl) Incubate samples at 95 C for 5 min.

3 Assembling the Gel System: 1. Remove the Ready Gel from the storage pouch. 2. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer. 3. Cut along the dotted line at the bottom of the Ready Gel cassette with a razor blade. 4. Pull the clear tape at the bottom of the Ready Gel cassette to expose the bottom edge of the gel. 5. Place one 16.5% Tris /Tricine gel behind the core and one buffer dam in front of the core. For each cassette, the shorter well side of the cassette faces in towards the buffer core. 6. Prepare 1XTris / Tricine / SDS buffer as described in buffer preparation section (2). 7. Fill the inside buffer chamber with ~150 ml 1X Tris / Tricine / SDS running buffer. Use enough running buffer to completely cover the sample wells. 8. Fill the outside buffer chamber with ~200 ml 1X Tris / Tricine running buffer. Loading samples 1. Use gel loading tips to underlay samples and marker into the gel wells 2. 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. Place the lid on the chamber and plug the lid into the power source. 2. Apply a constant voltage of 100 V for 100 min. 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 µg 1µg 100ng 100ng SBX1 SBX1 Marker SBX1 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.

4 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 PVDF membrane 2 second in 100% Methanol following 5 min in transfer buffer 3. Gently, remove the western blot gel from the plate using a tip or clean spatula around the gel. 4. Pre-incubate the gel in transfer buffer for 15 min 5. Place the cassette, with the black side down, on a clean surface. 6. Place one pre-wetted fiber pad on the black side of the cassette. 7. Place a two sheets of filter paper on the fiber pad. 8. Place the equilibrated gel on the filter paper. 9. Place the equilibrated membrane on the gel. 10. Complete the sandwich by placing a 2 pieces of filter paper on the membrane. 11. Add the last fiber pad. 12. Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the white latch. 13. Place the cassette in module. Repeat for the other cassette. 14. Add 1 liter of chilled transfer buffer to the tank. 15. Insert pre-freezed Bio-Ice cooling unit to the tank. 16. Place the lid on top of the buffer tank cell. 17. Connect the cables to the power supply 18. Apply a constant voltage of 25mA for 120min 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).

5 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 V5 antibody in blocking buffer. 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.

6 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