western blot ladder not transferring but proteins

Right now I have at least it down to some conclusions:ġ) the problem is not with my sds pH (even though it was at 7.5) or my tris lower buffer which i made the sds gels with because even if there were a problem with these it would only affect the running of the gel not the transfer itself, and since the gel ran fine, they should be fineĢ) Jacking up the voltage and extending the time makes my larger proteins transfer over better but as my small protein is still not visible either it diffused out or it's there but they are just simply very visible with ponceau stainingĭiffusion even of small proteins/polypeptides wanÂ´t happen in an electric field if their are propper loaded with SDS with NC itÂ´s the old problem of blotting through use PVDF instead if small and high molecular mass proteins/polypeptides should be on one blot I'm not sure if people typically see small proteins well on the membrane with ponceau staining. Now, since there is much less of the smaller proteins I am just hoping that it is there but I just can't visually see it with the staining because there is less of it. Well this time my transfer was better, I could see the protein bands much more distinctly except I still can't see my proteins at the 18kd size. In the meantime, if anyone has any suggestions please let me know! Thank you so much. Currently I am going to try at room temp at a higher V and longer time. However, on the other hand could it be the my protein passed through the membrane since my ladder has passed through? What could cause my ladder to transfer but not my proteins samples? Could it be the sds lower buffer (which I had to add a whooping 50ml of concentrated acid to adjust the pH to 8.8) or perhaps the sample buffer? since that is the only difference in treatment between the ladder samples and protein samples or the transfer conditions? I've tried it at 4 degrees O/N at 50V and at room temp for 1 hr at 100V and neither gave me good transfer. I've probed the membrane with actin antibody and saw very weak signals and since I see most of my protein left on the gel it seems I am getting an incomplete transfer. The strange thing here is that my ladder has actually completely transferred over and even passed through the membrane while my protein samples are all mostly stuck onto the gel. In a nutshell I've been having a great deal of problem transferring my proteins over to my nitrocellulose membrane for western blots. I have been discussing this issue under another topic forum that was originally addressing another issue so I thought it would be more appropriate to re-state the issue under this new topic.

Western blot ladder not transferring but proteins