I am a beginner in western blot and the results were okay for the last few weeks with beta actin as housekeeping control. The transfer buffer ran out a few days ago so I prep a new 10X transfer buffer with the following recipe:
Tris - 30.4 gm
glycine: 144.2 gm
Sds: 10 gm then make up to 1 L
For each Western blot I prepare fresh transfer buffer with 100 mL of 10 X transfer buffer and 200mL methanol and make it up to one liter with distilled water. But since then the results became very weird with faint and weak bands. The ladder and loading dye seemed normal in the gel but the bands were very very weak on the PVDF membrane. Ponceau stain after transfer reveal very very weak bands. I was wondering if there are any problems with my transfer buffer because it is the only variable. Also, the membrane seemed very dirty and grainy. Will these problems be due to the transfer buffer? Anyone has similar experience?? Thank you so much!!
Attached are the pictures of my results a few weeks ago and the membrane I got yesterday.
I don't think I can help you much. I am also struggling with western blot, but different problems. I just want to say that the recipe of transfer buffer I use does not have SDS. Your recipe above is exactly similar with TGS buffer (10X) in my lab. Therefore, my transfer buffer (10X) only has Tris-base: 30.3 g, and Glycine 144 g -> 1L and is filtered. Sometimes, my result is dirty and appear tiny spots due to the incompletely dissolved blocking solution.
I ll give my lab protocol for preparing 5X transfer buffer. it work well. Tris 75.75g, Glycine 360g , SDS 5 g. prepare up to 5 L. From 5X buffer prepare 1X transfer buffer by adding 400 mL pf 5X buffer, 200 mL of methanol and 1400 mL of DW. I guess maybe it is due to antibody problem. How did you dilute primary antibody? I usually used 1:5000 dilution of primary antibody in 5% non fat milk(TBST). incubate overnight. I used to block membrane by 5% nonfat milk in TBST. secondary ab too in 5% nonfat milk. I hope this will help for you. Good luck.
Thank you Ha Ngoyen!
We incorporated SDS in the transfer buffer because the size of our target protein is quite large. We do not normally filter it. Does it make a big difference?? It took a long time to dissolve for sure.
For the blocking solution, we use 5% skim milk prepared from skim milk formula. The formula has quite some history but it worked fine in the past as long as we let it settle for a bit.
Thank you and wish you luck with your western blot!!
Dear Thilini,
Thank you for your help. About the primary antibody, I also used the 5% skim milk like you but in 1:1000 dilution. I prepared the secondary antibody the same way as you. But I would like to ask if you use the antibody for a few times before diacarding or prepare it fresh every time? Thank you!!!
We use Tris-glycine buffer with 20% Methanol as transfer buffer without SDS and have no problem transferring proteins upto 400KD using SemiWet transfer at room temperature.
Christy Ng once dilute primary antibody, I use 3-4 times(Kept in -20 C) without any problem. I use beta actin primary antibody from Sigma.
Hi Christy! What system do you use for transfering? Wet or dry? I use wet and the transfer time varies acording to the weight of the proteins you are seeking. I use just 1 hour because my proteins are very little, but for large proteins you should use 2 hs or try other times. And if you are doing the transfer at 4C some of the SDS may be precipitating, so that's an issue.
In my opinion the source of variablility in this context is the experimentator.
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