I'm now doing western blotting for several proteins.
but transfer is not perfectly happened.
bands are looks like smeared or smudged.
And it seems to the lower MW bands are more affected.
what did I do wrong?
I use towbin buffer without SDS / PVDF 0.2um pore / biorad wet blot tank transfer system / during transfer, use stirring and ice block with 400mA (about 200~100V)
Also, when I performed with 20V overnight same thing happened.
I attached picture of my transferred membrane
when I stained membrane with Ponceau S solution, other protein bands looks same as marker band.
Do you have a photo of your gel? Does it run nice? How much protein are you loading per well? Are you boiling them before deposing them into the gel?
Hello Kim
Do you pre wet PVDF membrane with methanol before the transfer procedure?
When you set up the system for transfer see to it that the filter paper, sponge and the membrane are sufficiently moistened with the transfer buffer with no air bubbles trapped within. Carry out the transfer process for 1to 2 hours.
All the best.
If you are using Transfer buffer separately or ready to use membrane soaked in transfer buffer, make sure that they are not expired. If you are making the transfer buffer by your self to soak and wet the padding and membrane, use a highest-grade methanol (preferably a fresh HPLC grade methanol). I have noticed that the quality of reagents used for transfer buffer preparation often affects the quality of transfer from the gel to the membrane.
I hope this helps.
Jorgaq Pata Malcolm Nobre Pranav Joshi
thanks to all of your answers
but everything that you mentioned about is already I tried...
gel is nice, pre wet and equilibrated in transfer buffer for 10min, no air bubble, time 1hour to overnight, HPLC grade methanol used.
I think there's something I missing
I found last night may too much transfer buffer can be a problem.
I'll try one more experience today and I'll tell the result
gel picture attached.
Your gel is not so nice, you do not have resolved bands and you have a lot of smears. Put less proteins into your well, and denaturate them better (boil them or incubate at 75°C)? Is it a cell lysate or a membrane fraction of proteins? Is your gel migration buffer correct?
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