I am trying to analyse acetyl CoA carboxylase (ACC) protein (~265 kDa) using Biorad Criterion gels and Nitrocellulose membranes. I have already tried separating it on a 7.5% gel with 4% stacking gel, adding SDS (0.05-0.1%) to the transfer buffer and reducing methanol percentage to 10%. I run the gel at 100V for 2.0 hr and transfer on ice for 2.5 hr at 100V. Currently I am loading 10-20 ug of total cell protein.
The problem is that the transfer is variable and after stripping, there is no more protein left to do the loading control or the phosphorylated form of ACC. The lanes are distorted and the membrane is not quantifiable.
What else can I try to get reproducible and quantifiable transfer of this protein as well as enough protein so that I can strip the membrane and do the loading control at least?
I can't help you with your protein, but I work with a protein of similar size and I biorad, precast gels (4-15%), and run the gel for 3 hours, starting at 100v, then to 180 after 1 hour. Home made gels did not work well. Transfer is performed at constant amperage of 0.12A, and runs for 12-15 hours ( over night). I don't use constant voltage, because I find it over heats the gel. This process works on a few types of proteins running and transfer is done in a fridge,
Thanks Adrian I really appreciate your prompt response. I will try your suggestion. Do you use SDS and low Methanol percentage in the transfer buffer? And can you you tell me more about problems with pouring homemade gels bcoz I pour my own gels.
I'm not sure why the pre-poured gels do not work well for us on the large protein, but I guess I'm just lucky to get to use pre-cast gels. I use Bio-Rad precast gels cat no 456-1084. We make a 10x transfer buffer with 75g Tris 150g glycine in 1L water, then use it at 1x. You may need different conditions depending on the membrane you use and your protein
Hi
I think your transfer time is too long at 100V. Usually I just transfer at 80V for 1.5 hours.
One thing, You should use polyvunylindene difluoride membrane that easy to get strong banding with protein.
use PVDF membrane that it will help you to increase your protein transferring efficiency
I do transfer high molecular protein like fibronectin . For that 7% gel is fine for transferring i generally go for 300V and 25mA current for 2h and the transfer is proper. For transfer buffer 10% methanol is fine. Run the transfer in ice chilled transfer buffer. For further improving transfer you can use some proteases in the transfer buffer like pronase as this will helps in transfer of very high molecular glycosylated proteins.
Western blotting is a tool for qualitative analysis. It is at best a semi-quantitative assay. if your are expecting high levels of protein in your sample then set up a direct ELISA (or a dot-blot analysis) and use a pooled well-fined sample or if available a purified protein as standard. Then the data are much more reliable.
Otherwise, reduce the amount of protein applied per lane and use gel with large volume/lane e.g. 30µL/lane but still use/load the lowest amount of protein that still give good signals. This increases the area per lane and prevent clogging of the gel's mesh making the transfer more reliable.
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