Hello!
I extracted the protein from tissue, and trying to detect bands.
1) I am trying to see the specific bands for 4 antibodies, however,
non-specific bands at 50-60kDa is commonly detected for all antibodies on blots.
I heard that this is because that cell lysis is insufficient, is this right?
2) Also, I also heard that repeating freezing (-80C) and thawing could be helpful for cell lysis. Could repeating freezing and thawing cycle be more beneficial than applying RIPA buffer and homogenization? Sonicator is not available!:(
3) Protein ladder with lower sizes are not detected on X-ray film, and it was same on 10% and 15% gel. But I saw ladders with those sizes were transferred to PVDF membrane.
My protocol is
Transfer (100mA, 1h 30min-2h)
Blocking (5% skim milk, 2h, RT)
Primary antibody (diluted in 2% skim milk, Overnight, 4C)
Secondary antibody (diluted in 2% skim milk, 1h, RT)
I am a newbie for western blot, so It would be grateful if anyone could give me advice.
Thank you!
Hello Soo Hyeon
First and foremost you try using RIPA buffer rather than carrying out repeated freeze -thaw method. May be repeated freeze-thaw could have a damaging effect on the protein of interest.
Secondly try increasing the concentration of your primary antibody which should be of good quality. Also decrease your secondary antibody concentration. Usually secondary Ab dilution used is 1:10000 to 1: 20000.
The third point is too much protein loaded in the well could also cause non-specific bands. The protein concentration to be loaded in the well should be 10ug to 20 ug/well.
Also carry out thorough washing with wash buffer.
Thank you.
.
Kindly check for how to reduce non specific bands in western clot https://bitesizebio.com/26042/non-specific-binding-tips-to-sharpen-up-your-western-blot/
Also check https://bitesizebio.com/26042/non-specific-binding-tips-to-sharpen-up-your-western-blot/
Adjusting the concentration of secondary antibody worked for me!
Thank you all and have a great day:)
Hello Soo Hyeon
Nice to hear the news. You too have a great day and take care.
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