Hi Dave, I can't say it any better than Andrzej. Yes, it will
http://www.gbiosciences.com/PDF/Protocol/Ponceau-S_Stain.pdf
Sorry, this what of a rhetorical question. Spurred by a debate that I was having. The reason that I asked was because I have, as of yet been able to see any bands below 10kDa (green band) on a .45um PVDF membrane after 45, 30, or 15 minutes of transfer at 100V and 400mA using a 15% PAGE gel. The cell line was C6/36, a cell line derived from larval mosquito tissue. I am aware that most people recommend using .2um PVDF or nitrocellulose membranes, but I have seen other examples where this was not used (Vucic et al., 1997). Please see attachments.
Hi Dave, thanks for the clarification. To me, it looks like you don't have any major proteins running around 10 kDa.
Mind you, Ponceau staining is not very sensitive and there might be a protein there that will be recognized and stained using an antibody to your protein and augmenting the signal by using a HRP-labeled secondary and detecting the band using chemiluminescence.
HRP chemiluminescence detection of proteins can be >10e6 more sensitive than Ponceau S.
Dear Steingrimur,
Well thats been the problem. I've used three different antibodies for my 7.6kDa protein, all raised in rabbits then I apply HRP conjugated goat anti rabbit. For one antibody I get nonspecific bands, even in controls, that looks similar in character to the ponceau s stained membrane, with no bands below 10kDa. The other two antibodies didnt produce any bands. I use GFP as a control and it works great at the same dilution and ssecondary antibody (also goat anti rabbit) I get a clear band at around 27kDa. With no background. I'm beginning to think that the proteins lower than 10kDa are passing through.
Hi Dave, it certainly sounds like your protein is blowing right through the membrane without sticking.
You could try going with a 0.2 um membrane or do a double layer of the 0.45 um membrane.
http://www.emdmillipore.com/US/en/life-science-research/protein-detection-quantification/western-blotting/transfer-membranes/immobilon-psq-membrane/lOOb.qB.AfAAAAFB5JsRRkwz,nav;pgid=WZtMKfaYs81SRpEoh9VgbI7200007YgxuCoY;sid=WeMHHVxC15ALHQhvMjIXlbIX4GXFQgjgsWInTj9ARZJp0dQsgSFiV65eSFh-gnHbLbIpBI80mt7-teuOK8iM8yOnO0KDFNPH9zT8aXkWJdFN19gvvskdTYuyKSlc7VNyraVlvNhLmt7-tQjgsWInTj9A
That's interesting that you should mention immobilon membranes, I have actually looked at that same page before. Vucic and collegues actually reference another paper from that same year which indicates the method, they actually use the immobilon S membrane, which is .45um, and get good results, please see highlights in attachment (Vucic et al., 1997_)B. I have also tried using two (.45um PVDF) membranes sandwiched together to no avail when staining or using my antibodies. I just got some .2 um nitrocellulose membranes so I'm going to give them a try. Thanks for your advice.
Hi Dave, sorry I couldn't be more of a help, but please remember that many scientists only publish their best images and best results.
If the 0.2 um membrane does not work, you can try 0.1 um membranes:
http://www.sigmaaldrich.com/catalog/product/aldrich/z670766?lang=en®ion=US
Good luck!
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