Edit: image uploaded
I've blotted for beta-actin on cell culture protein lysates many times but the beta-actin detection range was not between 37 - 50 kDa range but 25 - 37 kDa range each time.
Details of the western blot: lysates are from primary mixed glial cells lysed in RIPA + protease inhibitors and stored at -80. Protein concentrations are read by BCA assay.
I do SDS-PAGE in a 4-15% tris glycine mini-gel , run them at 200V for 30 min. After washing in TBS-T, I transfer onto a PVDF membrane with the semi-dry transfer and easily see the bands on the blot.
Then, I block with 1% skim milk (NFDM) in TBST for 1h RT, incubate in b-actin antibody (sigma 1:10000) overnight at 4 C. Then I incubate with anti-mouse HRP secondary 1h at RT.
I detect the blot by ECL reagent from GE for 5 minute and capture it.
I did trouble shooting many times. I also blot the membrane with B-actin before anything else but I got the same result. You think the problem is with the protein amount or something else? I load 20 ug of protein and then try with 10 ug of protein but I got the same result.
Thank you for your answers!
Read the data-sheet of the antibody again.
Try running stacking gel at 50V for 30 minutes and 130 or 140V for resolving gel.If you are employing 200V throughout then you are not giving all the proteins appropriate time to align themselves and form a uniform front that they do in stacking gel and hence during separation you may observe actin at altogether different mol weight.
For the subsequent steps i would recommend to block in 5% NFDM instead of 1%
Hello there Ezgi Oz
When using 15% acrylamide gels we too see beta-actin closer to 37 kDa band rather than to the 50 kDa band on the ladder. However, at this % of gel the 37-50 range does not resolve very well, like the 25-37 kDa may (https://www.bio-rad.com/en-pt/life-science-research/overlay/precision-plus-protein-standards-migration-chart?ID=Dual-Xtra-_1278447005).
It would be helpful if you could put an image.
Your protocol seems fine, though 200V may be a bit much for the low% acrylamide regions, which may mildly collapse altering the overall protein movement. Nonetheless, if your ladder looks alright, it means your gel is fine as well.
Have previous work on your primary cells shown any specific modification of beta-actin. Sometimes lipidation causes proteins to run at slightly lower MW than their predicted MW.
Hope this helps
Best
Aparajita
Aparajita Lahree Thank you for your answer.
I will upload the image also.
My ladder looks alright although it might be missing the last two-three ladders which had 10 kDa to 20 kDa. This might explain what is going on.
I might also try to use lower voltage than 200 V and longer running time.
Greetings
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