I am a beginner of western blot and my target protein if 124kDA whereas the endogeneous controls are 41kDA and 55kDA. I did wet transfer at 100V, 80mins and successfully got the band for target protein.But both of my endogeneous controls did not show any band. I thought that it might be due to the overshoot of low MW proteins. Next day I cut the gel and did transfer at 100V for 40mins for the smaller ones but this time also there was no band.But in every cases ladder was perfectly transferred in the membrane. Can I transfer the target and endogeneous control at same duration and same voltage? Why do the low molecular weight proteins not show any band in western blot?. The blots are attached below.The first two blots represent controls.
Thanking you
Dear Maynak,
Are you using different primary antibodies for the endogenous proteins? Since you have mentioned that the ladder is being transferred well, I wouldn't be too worried about overshooting low MW proteins through the membrane. But in any case using a 0.22u membrane is always safe. Also a well labelled picture would help in understanding your question better.
Regards,
Abhishek
You need to be able to transfer your control and Target proteins together otherwise it is not an accurate representation of the transfer and you can't really use it to normalize your loading
What percentage of acrylamide are you using (I'm guessing around 8 to 10%?) and what membrane are you using for your transfer, nitrocellulose or PVDF? If you are using PVDF then you really do not need to worry about running the protein through the membrane since the pore size is typically so small, I regularly transfer 2.5 hours at 100V with no issue even for my 20KDa proteins, but I use a 12% gel. If you are worried layer 2 membranes on the transfer and process them together. If you are using PVDF make sure you are using MeOH to activate the membrane then transfer buffer to equilibrate.
Are your control antibodies well published and are you following the recommended BSA and buffer (PBS vs TBS) guidelines for the antibody dilution and blocking?
I know some people say you can incubate the primary Ab a few hours at RT but I recommend incubating ON at 4 C. Then 1hr at RT for the secondary An at the recommended dilutions
Dear Mayank,
You have to have always run and transfer your target awa control protein at same voltage and for the same time together always.If you are not seeing signal of your control protein only possibility is either you are not using proper dilution or proper incubation time. Or in the worst case your antibody is dead(which is least possible).If you are using actin or tubulin try using 1:1000 or 1:3000 dilution in 1X PBST with 5%BSA and 0.1% sodium azide with only 1-1.5 hr incubation at RT.
If your primary is not an issue then most likely check your secondary antibody.
Are you running 10% or 12% gel for this or something else?
Hope you get benefitted and get your result.
I think there are a lot of good suggestions already. Big and small proteins at least in the range you mention shouldn't be a problem in transferring. I often do 200kDa protein with alpha tubulin as loading control using 7.5% gel and nitrocelulose with no problems. I always check my transfer with ponceau, that helps getting a better idea how it transfered overall. I would go more for the antibody problems, check antibodies are ok and that the secondary is the correct one. Push it all to the minimum dilutions in order to see a band even if it burns the film and then optimize from there. Good luck
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