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Protocols

Western Blotting (WB)

Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas

Material

Method

  1. Prepare the running gel percentage according to the expected molecular weight of the antigen (Appendix I). Prepare the stacking gel and put the comb inside it. Put the gel into the tank.
  2. Prepare lysates by mixing with loading buffer and load as follows (in case of transfected cells put between 10-30ug/well, in case of no transfected cells or tissue extracts put 200ug/well).
  3. Fill the tank with the electrophoresis running buffer (Appendix II).
  4. Centrifuge the samples and heat for 5 min at 95º, centrifuge again and load into the gel.
  5. Use one well to load a molecular weight standard.
  6. Connect the tank to the power Pac Power supply and run the gel at 100 V until the gel front reaches the bottom.
  7. Wash the gel in blotting buffer (Appendix II) 10 min.
  8. Cut and wash the blotting membrane in methanol and in blotting buffer for 10 min.
  9. Prepare the blotting “sandwich” using the gel holding cassette. Remove the air bubbles by gently rolling a Pasteur pipette over the pad. Put it in the electrophoresis blotting module.
  10. Fill the tank with the blotting buffer (Appendix II) and connect it to the powerPac Power supply 2 hours at 200 mA (or overnight at 20mA).
  11. Incubate the membrane in PBS + 2.5% dry milk powder overnight at 4º or for two hours at 37º to block non specific binding to the membrane.
  12. Incubate the blotting membrane with the primary antibody for 1 hour at room temperature. Use another primary antibody as loading control (tubulin, GAPDH, lamin, etc).
  13. Wash the blotting membrane three times with PBS + 0.1% Tween 20 for 15 min each.
  14. Incubate with the corresponding secondary antibody diluted 1:2000 for 1 hour at room temperature.
  15. Wash the blotting membrane four times with PBS + 0.1% Tween 20 for 15 min each.
  16. Develop the blotting membrane with 5 ml of ECL for 1 min. Do it in darkness.
  17. Dry the blotting membrane with a filter paper to eliminate excess of ECL.
  18. Cover the membrane with a chemiluminescence film. Leave it for 1 min. Develop the film by using a developed machine.
  19. Observe the developed film and put another film covering the blotting membrane if needed.

Appendix I

Running gel
Acrilamide Percentage 7.5 9 10 12.5 15
0.75 mm gel 1 2 1 2 1 2 1 2 1 2
1M TrisHCl, pH 8.8 1 ml 2 ml 1 ml 2 ml 1 ml 2 ml 1 ml 2 ml 1 ml 2 ml
Acrilamide/Bisacrilamide 1 ml 2 ml 1.2 ml 2.4 ml 1.33 ml 2.66 ml 1.67 ml 3.34 ml 2 ml 4 ml
H2O 1.94 ml 3.88 ml 1.74 ml 3.48 ml 1.61 ml 3.22 ml 1.27 ml 2.54 ml 0.94 ml 1.88 ml
10% SDS 40 μl 80 μl 40 μl 80 μl 40 μl 80 μl 40 μl 80 μl 40 μl 80 μl
TEMED 5 μl 10 μl 5 μl 10 μl 5 μl 10 μl 5 μl 5 μl 5 μl 10 μl
10% PSA 15 μl 30 μl 15 μl 30 μl 15 μl 30 μl 15 μl 30 μl 15 μl 30μl
Stacking gel
Acrilamide percentage 3
0.75 mm gel 1 2
1M TrisHCl, pH 6.8 0.5 ml 1 ml
Acrilamide/Bisacrilamide 200 μl 400 μl
H2O 1.272 ml 2.544 ml
10% SDS 18 μl 36 μl
TEMED 2.5 μl 5 μl
10% PSA 7.5 μl 15 μl

Appendix II

10X Electrophoresis running buffer (SIGMA T7777)

10X Blotting buffer (SIGMA T4904)