Hello all,
I have run one SDS PAGE gel with 40ug of protein / well from mice adipose tissue. But I see some incomplete beta actin bands on my membrane. Please advice me.
Transfer method : Wet transfer for 2 hours at 65volts
Membrane type : Nitrocellulose membrane .45um
Primary antibody : Beta actin from Cell signal technology Rabbit origin (overnight incubation at -4)
Secondary antibody : Rabbit IgG from CST(one hour incubation at RT)
Blocking buffer : 5% NFDM in 1% TBST (one hour incubation at RT)
Please Find attached the image of the membrane for your reference.
Thank you,
Samnhita
Possibility
1: Voltage differences across the transfer area
2: Harsh washing of the membrane (but for b-actin it really doesn't matter)
3. 1% TBST is too much I guess: try 0.15%, Tween as a detergent tend to wash out antibody at higher concentration
4. Antibodies: I personally never used CST b-actin antibody. We get best result from SantaCruz actin antibody.
Hey Samnhita,
I agree with Vishal in points 1-3. It looks more like a transfer error or a problem with protein sample preparation (so before you wash and treat the mebranes with antibodies).
1% TBS-T is surely to much, we also use 0,1% Tween-20. But still in the case of actin it is very unlikely that you wash the first to bands of and the rest remains normal. We use Actin antibody from Sigma, but again, I think this is not the problem. Your bands look very crumbeld. How do you lyse and mix your protein samples before loading?
All the best and stay healthy,
Marc
Marc Herb and Vishal Khairnar
Thank you for your response. I did get an interrupted electric connection both during the running and transfer for this run. I usually use 0.1% tween in 1X TBS for washing the membrane.
Since those are the lysates of adipose tissue, I extracted protein by sonication and homogenization in 1X cell lysis buffer and then put all the samples in same concentration followed by adding blue buffer. Any kind of suggestion would be highly appreciated.
Thank you,
Samnhita
Marc Herb 1X cell lysis buffer + 1mM PMSF and 1% protease inhibitor for adipose tissue.
Marc Herb We use cell lysis buffer for adipocytes tissue lysates.
composition: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin.
Blue Laemmli Sample buffer for sample preparation
In addition to the previous comments, I think you might have had an uneven transfer unto your membrane. I would recommend you increase the voltage. 100 volts for 80 minutes or 2 hours (if you wish) should give you a very good transfer.
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