Hello everyone
I am trying to transfer a protein of 175 kDa on the Immobilon®-E PVDF membrane using transfer buffer (25 mM Tris, 192mM glycine, 0.1% SDS, and 20% methanol) at 100 volts for 120 min 4C. For the first time, I am using PVDF membrane (0.45uM); however, previously, I transferred this protein successfully on the nitrocellulose membrane (0.45uM) at the above-mentioned condition. Along with the above-mentioned transfer condition, I also tried 23 volt for 12 hrs at 4C for PVDF membrane. Unfortunately, this condition also does not work for my protein of interest. Please provide me guidance to troubleshoot the issue.
This sounds more like some error committed, like putting the membrane on the wrong side of the gel.
I have routinely transferred proteins of interest of 140 kDa (plus contaminants of higher and lower mass) using the following conditions:
- Gradient gels 5-15% T with stacking gel of 4% T, 10 mA const. current for the stacking, 20 mA for the separating gel. Under these conditions, you get good separation for proteins between 10-200 kDa.
- Prestained, multicolour molecular weight markers allow monitoring the progress of electrophoresis
- Dunn's transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, + 50 µM SDS, doi:10.1016/0003-2697(86)90207-1) works better and is cheaper than Towbin's. Soak the gel in this buffer for 15-20 min before transfer.
- PVDF membranes need to be pre-wetted with methanol, then with dd water and finally transfer buffer for a few s each.
- Proteins are given a negative charge by bound SDS; make double sure that the membrane is on the positive side of the gel. Paranoid people put membranes on both sides of the gel ;-)
- 40 V constant voltage for 1 h for transfer lead to complete transfer as proven by staining the gel afterwards.
- The prestained markers should be visible on the blot, reversible staining with Ponceau S makes other proteins visible (doi:10.1016/0003-2697(86)90263-0). The bands vanish again during the blocking step.
- For highly glycosylated proteins consider CTAB-electrophoresis instead of SDS (doi:10.1016/S0003-2697(02)00639-5, doi:10.1007/978-1-59745-542-8\_14) to get crisper bands.
You don't mention whether you use a molecular weight marker, as these will let you know whether the transfer is working. Ponceau S staining will help to visualise these.
(A) If you see the low molecular weight marker bands, but not the higher then the
transfer is slower than you anticipate. - increase the 100 mV run time to 4 hours - also check your transfer buffer composition.
(B) If you don't see any marker bands then there is something wrong with your setup, either the buffer, blots, or even the polarity are incorrect. The protocol needs checking as there is a mistake somewhere.
(C) If you see the full molecular weight marker ladder then something is wrong with your sample. Try replacing the sample buffer or exchanging the protein into a different buffer.
I completely agree with Professor Thomas Clarke . I believe he described it clearly!
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