Hi Khawla, I assume you ask because of the white band in your GAPDH in your third lane? This is most likely because of oversaturation. Loading less protein or using less antibody (either primary, secondary, or both) will most likely help with that.

Hi Khawla,

Have you validated your antibody?

Try to soak the PVDF membrane in Methanol for at least 5 minutes before soaking the PVDF in transfer buffer during transfer. This has helped me to avoid such scenarios.

Hi Simon Stuecheli

Good day, thank you for the reply!

Yes I was asking about that specific band

I usually load 40ug in 10-well gel

I'll start to troubleshoot the blot first if I get the same results I might load less than 40ug next trial.

Have a good day,

Hi Mohamed Khashan

Thank you for the reply!

well, I have not done this step yet, if you can send me a protocol I'll be pleased!

because I am also getting crossreaction with many other proteins.

Have a good day!

Hi Anupriya Bandyopadhyay

Thanks for the reply!

I am using nitrocellulose membrane instead of PVDF. I have once dealt with PVDF and it was necessary to soak it in Methanol to activate it.

Good job, Keep it up!

Have a good day,

Dear Khawal,

Pls refer to the following link where there are some tips related to your case:

https://www.bio-rad.com/en-tr/applications-technologies/western-blot-doctor-protein-band-appearance-problems?ID=MIW4Q8C4S#ghostprotein

Also: to decide on the sample amount that matches the antibody validation range:

1. Load different amount of your sample (10, 15, 20, 25, 30, 35, 40, 45, 50 ug).

2. Perform your protocol as usual.

3. Calculate band intensity and plot them against the sample concentrations.

4. You have to get an exponential line. When this turns into plateau phase, determine the corresponding concentration and use any concentration before it.

You have to take in your consideration that you use a concentration that fits to all antibody validation range.

Best regards.

Deaar Mohamed Khashan

Thank you, I'll do it InshaAllah

Have a good day!