Hello everyone. I have an issue with Western blot background when I use a higher percentage polyacrylamide gel during electrophoresis. The very same samples loaded into 10% gel lead to a very clear and nice blot.
In both cases I run the gel under 20 mA during stacking and 30 mA during resolving. I transfer for 90 min under 60 V and I use a nitrocellulose membrane.
Thank you in advance.
I would guess that the transfer is slower from the higher percentage gel, potentially leading to a lower signal due to incomplete transfer. This would reduce the signal-to-background ratio, making the background seem higher. If this is correct, a longer transfer time may improve the result.
Hi
In my opinion, the separating more proteins and many possible contaminants in the sample using a higher gel concentration could increase the gel background and affect the western results too. However,
I successfully used 14% stacking gel and performed the western blotting in 20V for 2-3 hrs using nitrocellulose membrane.
Using a higher percentage gel, such as 15%, can increase background because the smaller pore size can lead to less efficient protein transfer and more protein retention within the gel. This can result in more non-specific binding during the blotting process. You might consider optimizing transfer conditions or increasing the blocking efficiency to mitigate this issue.
Thank you all for your answers. For me, 2.5 h at 60 V seems to be optimal.
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