Flint Group Protocols

(3) How to do a Western Blot

Reagents

1.Protein quantitation kit. Available from Pierce. Product no 23227. Lot no EK65679. BCA Protein Assay Kit.

2. Hybond PVDF membrane. Protein Transfer membrane. PAK no RPN 303F. Lot no ND0030.

3.Resolving gels from Biorad. 12-14%. Ready Gel Precast gel system. 161-1101. 0.75mm.

4. Rainbow coloured protein ladder. Amersham. Code RPN756.

5. Secondary anti-mouse. Amersham biosciences. NA931V. Antimouse IgG, hourseradish peroxidase linked whole antibody.

6. 10x TGS running buffer. Biorad. 161-0772.

7. 10 x TG transfer buffer. Biorad 161-0772.

8. Western blot detection reagants. Amersham biosciences. RPN 2106.

9. Loading Buffer. Laemmli sample buffer. 161-0737. With beta mecap.

10. TWEEN 20 (Polyoxyethylene-sorbitan monolaurate). SIGMA. P-1379. Lot 18H0055.

11. PBS tablets. SIGMA. P-4417. Lot 093K8202. Phosphate buffered saline tablets.

12. Protease inhibitors. SIGMA P-8340.

13. ECL Western Blotting Detection. Amersham Biosciences. RPN 2106.

14. Polyacrylamide. SIGMA. A-3699, 30%.

15. APS. SIGMA. A-3678. 25g.

16. Temed. Available from BDH, Electran.


Method

Lysis

  1. Lyse tissue. This requires a lysis buffer: 50mM Tris-HCL (ph7.4), 150mM NaCL, 1% Triton x100 and protease inhibitors. Always make up the lysis buffer from fresh, as it contains protease inhibitors that have a limited shelf life. These PIs are kept in a plastic bag in the top compartment of Dave’s Freezer. To make up 30ml of lysis buffer add the following in a 50ml Falcon:

-  1.5ml of 1M Tris-HCL

-  4.5ml of 1M NaCL

-  300µl of Triton. (Add this last and dissolve using hot water)

  1. Place lysis buffer on ice. All lysis steps should be done at 4oC. Add requisite amount of protease inhibitors (they come in 10µl aliquots). Rinse out ‘every last drop’ of PI in the tube using lysis buffer. Make up the lysis buffer to 30ml by simply hand pouring MQ water to the 30ml mark on the Falcon.
  2. Add 1ml of lysis buffer to large brain regions such as the cortex. Add 100µl to regions such as the hypothalamus, or superior colliculus. Most brain regions can be lysed in 1ml. If homogenizing spleen, one half of the organ should be sufficient, with enough lysis buffer to submerge the tissue, say 300µl to begin with. For bone marrow samples, add 100µl of lysis buffer to Epp.
  3. Homogenize samples. Sample should be kept ON ICE as far as possible. This can be done with either a pellet pessel or a homogenizer. For spleen samples, we use a 3ml glass mortar and pestle to grind tissue down, again done on ice. Then transfer homogenized spleen to an Epp. For bone marrow samples, vortex rapidly after adding 100µl of lysis buffer to Epp. Then spin down. You can use Dave’s pellet pestle motor to attempt to breakdown any remaining tissue (i.e. harder marrow), but transfer sample to a special blue tube first.
  4. All tissue to lyse for 1hour on ice. Vortex intermittently.
  5. Centrifuge at 20,000g (or max) for 30mins at 4 oC.
  6. Take off supernatant, avoiding particles, and pellet.

Quantitation

  1. To quantitate the protein use the BCA kit. Begin by diluting sample 1/20 in a total of 50ul of water.
  2. Prepared the BSA standards. Do a serial dilution, for best results. Remember a blank, just with 50ul water. Make up standards fresh each time.
  3. Prepared BCA mix. 50:1, solution A: solution B. eg 10ml mix with 9.8ml solution A, and 200ul solution B.
  4. Add 1ml of BCA mix to 50ul samples.
  5. Incubate at 37 degrees for 30mins.
  6. Allow samples to cool and measure on spectrophotometer at 562nm within 1hr.
  7. Create a standard curve using Microsoft excel, and calculate protein concentrations.
  8. Prepare samples to load on gel.10ul sample, and 10ul loading buffer.
  9. Vortex and spin down samples. Denature at 100 degrees for 5 mins.

Making a Gel

  1. Wash gel plates with ethanol
  2. Place gel plates in green rack, and load onto plastic rack.
  3. Prepare resolving gel. For 10% resolving gel (3.65ml water, 3.75 1M Tris-HCL ph 8.8, 100 ul of 10% SDS, 2.5ul of 30% acrylamide, 50ul 10 % APS, 5 ul Temed).
  4. Make up APS fresh (0.1g in 1ml of water).
  5. Add temed last.
  6. Prepare gel for loading. Place combs in gel, and mark just below combs.
  7. Fill gel with pipette to marked line.
  8. Add water while resolving gel sets.
  9. When resolving gel is set, pour of water and add stacking gel (3.8ml water, 625ul of 1M TRIS-HCL ph 6.8, 50ul 10% SDS, 500ul of 30% Acrylamide, 37.5ul APS 10%, and 7.5ul Temed).

Gel Running

  1. Load gel, and run at 120V until end.
  2. Run gel in running buffer from BioRad, 1x. Make sure it does not leak. Inner compartment should be full of buffer, and some at the bottom as well. Does not matter if entire tank is full with buffer.
  3. After gel has finished running equilibrate in Transfer buffer for 10mins. Remove first glass piece, but let second piece remain there.

Transfer

  1. Make transfer buffer (100ml of TG, 200ml of methanol, 5ml of 10% SDS in a total of 1l, rest water).
  2. Prepare whatman paper (7cm x 9.5cm) and PVDF membrane (6cm x 8.3cm). 8 pieces of whatman paper are required per a gel.
  3. Activate membrane for 10 secs by placing in methanol.
  4. Equilibrate activated membrane in transfer buffer for 10mins, rocking.
  5. Put membrane on gel, and make sure no bubbles.
  6. Get transfer apparatus. Moisten sponges. White side closet to you. Place sponges, 4 pieces of wet whatman paper (place in transfer buffer) on white side.
  7. Put gel and membrane into sandwich. Membrane should be closet to white side, and get towards black side.
  8. Add another 4 pieces of whatman paper and sponge. Sandwich. Place in transfer apparatus (black on black).
  9. Transfer overnight at 50volts, at 4 degrees. Alternatively 1.5hrs at room temp with icepacks in apparatus at 200mA. Be sure to have a flea, and metallic stirrer going so the transfer buffer does not stagnate.

Blotting

  1. Prepare blotting buffer (0.2% Tween 20 and PBS). For 1L, add 5 tablets of PBS and 2ml of TWEEN 20.
  2. Take 100ml aliquot of this, and add 5g of milk powder for blotting.
  3. Remove membrane from transfer apparatus. Place in weighboat and add PBS/tween with milk powder, (about 50ml per membrane) and rock gently for 1hr (or 4 degrees overnight). This blocks the membrane.
  4. Incubate membrane with primary antibody for 1 hr (alpha tubulin 1:1000 dilution, mouse monoclonal antibody, T6199). Dilute antibody in PBS tween.
  5. Wash membrane for 3x 5min in PBS/TWEEN.
  6. Add secondary antibody (2.5ul of antibody into 25ml of PBS/TWEEN, 1:1000) incubate for 1hr at room temp, with rocking.
  7. 3 x 5 mins washes in PBS TWEEN.
  8. Make up 3ml of ECL detection mix per a membrane.
  9. Dry membrane by dabbing on tissue. Place membrane on plastic film. Add 3ml of detection mix. Incubate for 1 min. Tip off ECL and wrap in new plastic film.
  10. Visualise on film.

Stripping

  1. Stripping of the membrane allows you to re-probe with another antibody so you can make sure loading is consistent.
  2. Make up stripping buffer (100mn B-mecap, 2% SDS, 62.5mm Tris HCL ph 6.8). For 500ml of stripping buffer: 3.5ml of 14.3M Beta Mecap; 50ml of 20% SDS; 31.25 1 M Tris-HCL ph 6.8 and 415.25ml of water.
  3. Prewarm stipping buffer at 55/60 degrees for 10mins
  4. Incubate membranes for 30mins at 55/60 degrees.
  5. Wash three times with PBS/Tween using large volumes.
  6. Reblock with milk powder and probe.

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12.02.08