NOTE: This protocol is NOT applicable to all Western blots. It has been optimised for the detection of myostatin, out of the chemicals and products that are mentioned in the protocol. I am not guaranteeing that it will work for your protein. I am just sharing this with you so you can see what a protocol may look like, and construct your own out of it.

 

 

            Protocol for detection and quantification of myostatin

 

1.   Homogenise and prepare the tissue. Determine the amount of protein in the samples, using a suitable protein assay.

2.   Denaturate the sample with Laemmli Sample Buffer/DTT/IAA (Bio-Rad Laboratories/BDH Biochemical/Bio-Rad Laboratories) according to protocol. 

3.   Fasten the gel (CriterionTM Precast Gel 4-15% Tris-HCl, Bio-Rad Laboratories) in the electrophoresis tank and fill up with running buffer (10x Tris/Glycine/SDS pH 8.3, Bio-Rad Laboratories) diluted 10 times (200ml buffer + 1800ml ultra pure H2O à 2000ml). Apply standards (2μl Cruz MarkerTM Molecular Weight Standard, sc:2035, Santa Cruz Biotechnology, Inc., 6μl Kaleidoscope Prestained Standards, Bio-Rad Laboratories) and 12μl denaturated sample to the wells according to the following scheme:
blank – Cruz MarkerTM – sample 1 – sample 2 – sample 3 – sample… – Kaleidoscope

4.   Run the gel for 50 minutes, 200V, or until the Kaleidoscope colours are fully separated.

5.   Pre-wet the nitrocellulose membrane (Bio-Rad Laboratories), 4-6 filter papers/gel and 2 sponge pads/gel for at least 20 min in transfer buffer (100ml 10*transfer buffer + 200ml methanol + 700ml dH2O. 10* transfer buffer = 30.28g Trizma® base (Sigma) + 144.1g Glycine (J.T. Baker) + dH2O to a total volume of 1l.). The membrane should be placed on a shaking plate.

6.   When the electrophoresis is completed, let the gel soak on a shaking plate in transfer buffer for 20 min. 

7.   Make a “sandwich” by piling the following layers, in order, onto the black side (- electrode) of the cassette:
sponge pad – 2-3 filter papers – gel – membrane – 2-3 filter papers – sponge pad 

8.   Place the sandwich in the transfer tank together with a magnet and a cold clamp. Place the bucket on ice in a cold storage room and run the transfer at 100V, 60 min.

9.   After the transfer, wash the membrane 4*1 min in TBS (10*TBS = 24.22g Trizma® base + 80.06g NaCl, pH 7.6. For 1*TBS, dilute 100ml TBS with 900ml dH20.).

10. Stain the gel with Coomassie Brilliant Blue (0.2 % Coomassie Brilliant Blue, 40 % methanol, 7% Acetic acid, 53% dH2O) for at least 15 min before destaining (40 % methanol, 7% Acetic acid, 53% dH2O).

11. Pre-block the membrane in 5% blocking buffer (1*TBS + 5% dry milk (Semper) + 0.1% Tween®20 (Bio-Rad Laboratories), pH 7,5). Incubate on a shaking plate for 60 min.

12. Dilute primary antibody, GDF-8 (C-20): sc-6884 (Santa Cruz Biotechnology, Inc.): For 1: 5 000 dilution, add 6μl antibody to 30ml 1% blocking buffer (1*TBS + 1% dry milk + 0,1% Tween®20 , pH 7.5). Make sure the components are well mixed.

13. Pour away the pre-block solution and apply the primary antibody solution onto the membrane. Incubate on a shaking plate (at a low rpm) in a cold storage room over night.

14. Dilute secondary antibody, donkey anti-goat IgG-HRP: sc-2033 (Santa Cruz Biotechnology, Inc.): For 1:10 dilution, add 2μl antibody to 18μl 1% blocking buffer (1*TBS + 1% dry milk + 0,1% Tween®20, pH 7.5). Add 6μl of this solution to 30ml 1% blocking buffer. This results in 1:50 000 dilution. Make sure the components are well mixed.

15. Pour away the primary antibody solution. Wash with TBS+Tween®20 (1*TBS + 0,1% Tween®20) 2*1 min and then 3*5 min. Let the washings take place on a shaking table.

16. Apply the secondary antibody solution onto the membrane. Incubate for 60 min at room temperature on a shaking plate (at a low rpm).

17. Wash with TBS+Tween®20 2*1min and then 3*15 min.

18. Wash with TBS 4*5 min.

19. Dress the development cassette in plastic foil.

20. Mix the two solutions of the ECL (enhanced chemiluminescent) Plus Western Blotting Detection Reagents (GE Healthcare, formerly known as Amersham Biosciences) according to protocol, i.e. 40:1 (for example 20ml of reagent 1 and 500μl of reagent 2).

21. Incubate on a shaking plate for 5 min.

22. Let excess liquid drop off the membrane. Place the membrane in the cassette and cover it with another layer of plastic foil.

23. Develop in a Curix 60 developing machine (Agfa) according to product instructions.

24. If the amount of protein in the bands is desired, density can then be measured by using a Chemidoc™ scanner (Bio-Rad Laboratories).