Dear All,
I just wonder that is possible that some protein might not transfer to some type of membrane in Western Blot. for example protein A might transfer to only nitrocellulose, not PVDF.
I have a case that I and my colleague using the same cell line, same protocol, and same antibody to detect different isoform of calcineurin A at 48kda and 60kda. I use PVDF and detect only 60kda, but my colleague use nitrocellulose and detect both 48kda and 60kda.
I'm still thinking what is the issue. The pore size of both membrane are the same 0.45um, so it couldn't be issue. Usually larger molecular weight transfer to membrane more difficultly. But in my case, 60kda has alreaday transfer, but not its lower MW, 48kda doesn't. PVDF binding capcity should allow more protein transfer. Ah I dont know.
Thank you
Cheerio,
Yes,
binding properties of PVDF and nitrocellulose differ significantly. Hence, given protein can be trapped with markedly different effectivities by nitro or PVDF. You need to optimize it - as everything in WB:-(
Martin
There are plenty of differences (I would recommend you to google it). As a main difference I consider hydrophobicity (PVDF)/hydrophility(nitro). However, it is hard to predict, which protein would bind preferentially to which membrane. We typically try both. But some researchers work with PVDF only, some prefer nitro (chemiluminiscent detection on nitro usually works better). When I am lazy to try both, I start with nitro, unless the protein is small in size (under 10 kDa).
Asa, is there any possibility that you should not have the 48 kDa band? I tell this because I find strange that you see one of the two.
Different membranes have different properties; also Western with one membrane but trying to look at two proteins with two diferent sizes may be a problem. But to see a 60 kDa and not see a 48 kDa is strange. Probably it is not there (or is there but at much lower concentration), or may be you have problems in the transfer in this part of the blot. The latter would not be due to the use of a different membrane but to a problem in the blotting set up.
In any case my recommendation is to do a general protein stain after blotting in which you will see if the blotting was correct and took place homogeneously in all MW zones.
Best
Rafael
Hi Asa,
Rafael points out some very important aspect as I think 48 and 60 kDa are not that very different to be seen on a PVDF membrane, assuming you have optimized every thing else for a WB.
Have you tried 'your own samples' on a nitrocellulose membrane and did you see bands for both isoforms? if you have not done yet, its worth to try before you do any more other analysis.
Good Luck
Venu
Thank you all,
I've googled it about PVDF and nitrocellulose, both binding interaction are hydrophobic. So at first I thought the membrane wasn't the problem. But, as Venu suggested, I think I will try it on nitrocellulose.
As Alim and Rafael mentioned, it could be transfered problem. I thought about this too and I didn't stain membrane after the transfer, but biotin protein marker all present from 120 to 10kda. So I think it is not transfer problem.
Yes, try it on nitro. I am quite sure, this is the trouble. For some proteins, we have repeatedly seen no signal on PVDF and very trong signal on nitro (done within one experiment under the same conditions). Martin
Several of the answers have alluded to a variety of issues that need to be considered when you start getting odd Western results. I have found that empirically testing different conditions FOR EACH PROTEIN OR BAND may be required. There is no "one condition works for all proteins". Things to consider include, the voltage and length of time of transfer, the % acrylamide and ratio of bis:acrylamide of the gel you are transferring out of, the blocking solution, the temperature at which you transfer, the length of time you incubate the filter with primary antibody. I have found each of these to contribute to confusing results over the years.
Finally, how do the two size bands you are looking for differ? Are they splice variants? Is one a degradation product of the other or post-translational modified?
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