5 replies to this topic

[Dave_Kub_11]

Hello everyone,

I recently moved to a new lab and my PI and I have had problems with our Westerns.  The problem seems to begin with the running of the rainbow marker.  Around 50kDa it becomes extremely difficult to see in the gel tank but the dye front of the protein is still easily visible and runs with no problem.

Upon Ponceau staining after transfer, the proteins appear to run fine until they reach around 40-50kDa then it appears as if there has been no transfer of proteins.  I have a pic to demonstrate:



This is from a 12% gel where I intend to probe for Histone 3 which is around ~17kDa.  However, there appears to be no transfer below ~50kDa yet the markers come over suggesting it is more of a probelm with the gel run.  I have routinely blotted for this protein on numerous occasions and have never encountered this problem before.

I have tried fresh reagents and even went so far to re pHing the buffers etc.  My colleague is convinced it may be a problem with the water as she has had problems of this ilk too.  I even switched from autoclaved water to filtered milli-q water but that made no difference.

Any suggestions would be greatly appreciated.  Many thanks.

[ascacioc]

It is a problem of the running buffer; most probably it contains the double amount of glycine. Make a fresh one.

[Dave_Kub_11]

 ascacioc, on 10 October 2012 - 08:58 AM, said:

It is a problem of the running buffer; most probably it contains the double amount of glycine. Make a fresh one.

I usually dilute a 10x stock of running buffer which contains 140g Glycine, 30g Tris-Base, 0.5g SDS per 1 litre.  Never had a problem before with the running buffer.

[crom80]

I too am having similar problems. After Ponceau Staining I do not see stong bands below 50kDa. My molecular weight markers look fine. So I thought it was transfer issue but when I Coomassie stained my 12 Gel prior to transfer I did not see stong bands below 50kDa. Still my markers look fine. So I am currently thinking it my be the electrophoresis rig problem or gel polymerization problem. I will update.

Edited by crom80, 15 November 2012 - 10:53 AM.

[mdfenko]

if the standards ran well then it is most likely not something with the gel or buffer. it is most likely something with the sample. since all of the samples are running similarly then it is likely due to the buffer that the sample is in or the sample loading (laemmli) buffer. is the sample buffer old? the sds may be decomposing.

try making a fresh sample loading buffer (use a different lot of sds).

[crom80]

 mdfenko, on 16 November 2012 - 11:01 AM, said:

if the standards ran well then it is most likely not something with the gel or buffer. it is most likely something with the sample. since all of the samples are running similarly then it is likely due to the buffer that the sample is in or the sample loading (laemmli) buffer. is the sample buffer old? the sds may be decomposing.

try making a fresh sample loading buffer (use a different lot of sds).

My problem was fixed by making fresh sample buffer. Thank you.