I am running a 12 % SDS gel for my protein expression (targets are between 35 - 17 kDa). I use a mini biorad setup for gel preparation. I run at 60V for 30' followed by 100V for 1 hour and 15 minutes (until I see the front dye at the bottom - indicated with an arrow on the picture attached). For transfer, I do it in an ice box at 100V for 75 minutes.
I use Tris Glycine Running buffer with SDS.
For transfer, I do it without SDS (Methanol included).
Is there a chance I am loosing small proteins due to my prolonged running and transfer?
attached picture is for the PVDF blot probed with Ab for IL1b (I do see a lot of non specific bands but not really a specific one - I will be optimizing blocking and dilution for my Ab).
Samples were prepared with RIPA.
You can see from the marker protein ladder the size range of proteins that are separated in the gel. If the marker dye is still on the gel after electrophoresis, smaller proteins will most likely be located at the dye front. If the dye has run off the gel, the smaller proteins will have run off and will be lost.
Small proteins may be lost during blotting if the blotting time is too long.
Hi,
I don't think your transfer protocol was a longer one.
What is the mol.wt of the lower band for the marker and the non-specific band shown in your blot? Could you please label the molecular weight for the standard
Hi Fatima!!
If you're targeting proteins smaller than 20-25 kDa, I recommend blocking the membrane (whether PVDF or nitrocellulose) with 4% PFA (some use 0.4% as well). Simply apply 4% PFA to the membrane, focusing on the side with the brighter ladder bands, and incubate for 15-20 minutes at room temperature. After incubation, wash the membrane once with 1X PBS, then proceed with blocking using 5% skim milk, following the standard protocol. This additional PFA blocking step can help prevent small monomeric proteins from washing away during subsequent washing steps.
For optimal results, limit your washing steps to 3 cycles of 5 minutes each. I hope this improves your Western blot results. Best of luck, and feel free to share your experience!
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