Strange problem with actin control in Western blot
Posted 02 February 2005 - 02:32 PM
I have been using beta actin as my internal standard to ensure that equal amounts of protein is loaded. But the problem is, i could detect a very clear band only in one out of the ten lanes on the membrane. I could detect my protein of interest in all the lanes. I am stripping the membrane after i used it for my antigen of interest. Could any one tell me why i am unable to detect actin bands in all the lanes?
This is urgent!
Any help is greatly appreciated.
Posted 02 February 2005 - 09:53 PM
Let me explain my problem more clearly. I ran 5 control samples and 5 diseased animal samples in a 10 lane gel. I am specifically looking for GPCR transmembrane receptors. I am getting good bands for those receptors. But i am able to see a band for beta actin only in one lane (5th control sample). And this band was really intense and clear. But i could not see even a shade of a band in the other 9 lanes. I use a reducing loading buffer with beta-ME and camiolo buffer for lysis and homogenization. As far as i know i didnt give any special treatment for this sample for which i got the band. What could be the possible reason for this paradoxical observation? What could have caused the disappearance of the bands from rest of the lanes?
Thanks in advance.
Posted 03 February 2005 - 04:19 AM
i cant think of anything.
but from my experiance, i HATE striping membranes. i find a stripped membrane never works as well as a fresh one. maybe that was the problem, u may have stripped for too long or something.
But i do have an idea, to help you isolate the problem. Your protein is almost certainly larger than 75 kd, correct?
So, after transfer, before you wash away your ponsoue, use the red marking and cut your membrane just under the 75 or over the 60 Kd marker.
then you do not need to strip to blot for actin. just add to the lower membrane anti-actine and the other part, your antibody.
Edited by Zaax, 03 February 2005 - 04:21 AM.
Posted 04 February 2005 - 09:36 AM
thanks for the reply. Actually, i usually get the band of my receptor of interest at around 45 Kd. So probably its little difficult to follow what you suggested. However, yesterday i ran the gel again and tried with actin first (i.e no stripping). I got bands at ~80Kd, almost double the size i expected. Perhaps actin is forming dimers. Today i am running another gel with samples boiled. Let me see how it works.
thanks again for ur suggestion .