Maybe/sometimes it is the truth. IP is kind of the process of purification and enrichment. so the signal is stronger. Did you run Western with total lysate before IP? Thanks.

Thanks for your response. Yes. the one I labelled was total lysate before IP and the bound after IP

How specific is the antibody? Does your protein form dimers/trimers/tetramers? Are there glycosylated/phosphorylated residues?

First, you must measure the amount of protein in this sample and compare it with the first samples to make sure they are equal.

Secondly, the Albertine sample must be analyzed using ًzone electrophoresis@ and compared with normal samples.

@ Katie,

Thank you so much for your response. Sorry for my late reply. My protein can form dimerization. The antibody I used for my experiment was streptavidin coupled HRP since my protein tagged with Biotin. I have verified in the uniprot that my protein has phososerine modified residues after PTM. Do you thing this properties affects the protein running on the gel? Moreover, I never had this problem before when I use the same protein with different tag.

@ Yacer,

I have loaded the equal amount of protein in the gel. Sorry I forgot to mention in the questions. What you are referring albertine sampe?

Thank you for your response.

1. "Equal amount", either equal ng or equal uL does not mean equal POI in loading and on the membrane.

2. Dimer: You may need to treat both "total lysate" and "bound protein" with the same method, denature or non-denature.

3. Biotin: May try blocking with BSA, not defat milk, if I remember correctly.

4: Smear: The POI antibody may not be specific and not perfect, or the blocking.

Feng Wang

Thank you for your detailed response. I have loaded 1% of cell lysate and bound from the total volume. I have denatured both lysate and bound protein. Since its a Biotin tagged protein, I have used BSA for blocking.

Thank you all for your immediate response. I would like to share the western blot image of serial dilution of my protein. So, I found that due to the vast number protein in the cell lysate, the antibody couldn't detect the protein in the cell lysate. When I load the different dilution of my cell lysate, I could see the expression clearly.

Thank you all for your feedback and explanations.