Strange pattern after transfer - (May/10/2009 )

Hi all,

I have been using semidry transfer for quite some time now and had no problems so far. Results were always good and with minimal background. One month ago i bought some new Abs for my protein (32 kD) and since then I am having very different patterns of results. Most of the time I will get extremely thin bands, look almost like hair and I dont know where this comes from. Now, i have looked into various things that can interfere but one thing that I cant figure out is whether the blotting pads make problem or not. I must say when I had the good results I was using blotting pads from VWR that are thick and in the meantime that I run out of them I was using some other ones from BIORAD that are a lot thinner and I have to use multiple of them for one blot.
Could this be the reason? Does anybody have any experince with this or am I loosing my sanity?

Thank you so much

The thickness of the pads shouldn't be a problem, we just use cheap scouring style cloths from the supermarket. So long as they are holding the gel to the membrane firmly enough it will be fine.

I think you need to check your buffer composition and pH - storage of transfer buffer can give some very funny results. You may also want to look at your running buffer, often things that go wrong are an incorrectly made running buffer, the wrong pH can cause the proteins to migrate oddly.

bob1 on May 10 2009, 05:57 PM said:

Hey Bob, thx for your answer. The transfer buffer we use is always a fresh made buffer (Towbin buffer with 20% MeOH). The running buffer is a 10x stock. The transfer buffer doesnt need titration cause it is just Tris, Glycine and MeOH and dH2O. I will check the running buffer, but we must destinguish between migration and transfer. The Ponseau S gives me very good bands always, so i assume the transfer works. The problem is concentrated on the pattern I get after developing the film. Shall I upload you an image? maybe this will help

If your ponceau is fine, then the run shouldn't be the problem. Is heat a problem with the transfer unit? Sometimes that will result in funny bands too. 32 kDa is reasonably small so there shouldn't be a problem with the transfer, unless you have some problem with the blot unit. Could you try running another antibody over the blots? A loading control Ab would be a good test - something like beta-actin or alpha-tubulin. Does this give you the same odd bands?

bob1 on May 11 2009, 06:34 PM said:

Hey Bob,

thx for your answer. I tend to suspect my sample. Maybe my protein is degenerated cause in the small Eppendorf tube I keap it there is a small precpitate on the bottom. I run today a sample from a fellow that I know that his protein is good and I will test in this way my own way of doing WB. I will get back to you soon with the results. If i still get that odd result then i will runf the test Ab to see what is happening.
By the way the Ponceau S gave me today very good bands so I assume that in the sample i tested today there is indeed protein

Is your antibody polyclonal? Is it a new batch?
It's a good idea to test other samples. Do you still have one old frozen sample that worked fine?

little mouse on May 15 2009, 01:12 AM said:

so I am back and I have tested some other proteinwith another Ab and i tend to think that my protein was bad since the blot today gave me excellent results. Yet I dont really understand why the BCA gives me a valid measurment? as if i have indeed protein in my sample. Or is it just fragment of AS that react and give the color that the BCA method is tracing...

The BCA will work on short protein fragments so long as there are bonds between them to reduce the Cu²⁺ to Cu¹⁺... so it will work on degraded samples.

bob1 on May 17 2009, 05:31 PM said:

Thx everybody for the kind replies. I run a new sample of newly isolated protein and found out that the former protein i tested was not good.
I usually leave the cells on ice after i have lysed them for ca. 15 min. Probably i have left them way too long that time and the protein degenerated.