Protein samples not transferring over to membrane - Western Blot (Oct/03/2006 )

Hi all,

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. In a nutshell I've been having a great deal of problem transferring my proteins over to my nitrocellulose membrane for western blots. 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. 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.

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. Currently I am going to try at room temp at a higher V and longer time. In the meantime, if anyone has any suggestions please let me know! Thank you so much.

My 1X final SDS sample buffer is as follows with 5% B-me added:
62.5mM Tris HCl, 2% SDS, 10% Glycerol, 0.005% bromophenol blue, pH = 6.8

SDS in your transfer buffer can impede proteins from transferring to nitrocellulose...Bio-Rad recommends only methanol in transfer buffer for nitrocellulose. But I don't know if that's the issue for you.

Do you use prestained reference proteins? It may be the dye that was transfered without proteins; also, the dye alone can simply be blotted through

SDS should be no problem as proteins should carry enough of it; as suggested you can blot without but a small amount (0.1 %) is ok;

I see only 3 things:

gel/proteins were fixed before blotting (by what reason ever)

PVDF was not activated (with methanol)

current was too low

WAstate, do you have to adjust the pH of your sample buffer? I think our was made was Tris-Cl 7.5 rather than 6.8 but the gel ran fine so it shouldn't be a problem. Kosmodrom,, I use a nitrocellulose membrane so it doesn't need methanol activation and I had stained my gel and saw that the ladder proteins did not remain on the gel and so it must have blotted. I am currently running at a higher voltage (125V) at room temperature for a longer time to see if that would do anything. Thanks for all your suggestions! I will update on the results....

I highly doubt 6.8 vs. 7.5 would matter at all. I don't make the buffer because I just buy the lysates ready to load on the gel, but I do add the B-me, so I AM doing some work

Haha I see,

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. 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. I'm not sure if people typically see small proteins well on the membrane with ponceau staining. Right now I have at least it down to some conclusions:

1) 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 fine

2) 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 staining

tell me if that makes sense?

Hi there

Can I ask the size of the large protein that you had difficulty in transferring? I am currently having the same problem (my protein is around 65-70kD) and thinking about increasing the voltage or transfer time.

Hi lil,

The protein which I am having trouble transferring is actually very small, around 18kd. But initially I was having trouble transferring over even the larger proteins and currently I have solved that problem by extending the transfer time. I did it at 100V at room temperature for an hour. Also, be sure you add SDS to your transfer buffer, I use 0.04% but up to 0.1% can be used. I tried it without SDS before because I was worried my small protein would pass through the membrane and didn't get a transfer at all! I still haven't gotten my smaller proteins to transfer but for a large protein like yours, increasing transfer time should work.

kosmodrom,

About the diffusion problem, I was thinking perhaps soaking my gel after I ran the sds page and before i do my transfer would have caused the smaller proteins to diffuse out? I heard for small proteins we should not soak the gel in transfer buffer for such a long time, esp. since I am using a mini-gel. I had soaked my gel for quite a bit like 20 minutes so there's a chance the proteins could have diffused do you think?

mdfenko... I do use a 0.2um pore but thanks for the suggestion!