hi

It could be because of few reasons such as

1. The chemiluminescent reagent hasn't worked.

2. Protein may not be available in the blot to react. This could have happened because of few obvious reasons like

a. transfer has not happened properly.

b. the secondary antibody didn't work.

c. primary antibody didn't find antigen. May be protein got degraded or the SDS gel was resolved too much and the interested protein has gone out. Well, this can also happen if you resolve and cut the blot according to the size of proteins to detect multiple proteins from one PAGE.

Regards

Saurabh

Hi Gareth Rostro

It would be helpful to add images of interest.

Beside to was mentioned before. Too high concentration of either Primary or secondary antibody, insufficient washing, membrane drying, too high volume of chemiluminescent or rolling on the membrane by dirty roller during blotting sandwich construction can lead to dark membrane.

Best....

Saurabh Mandal thank you so much for you rewarding help.

Your shares has made me think about the state of my proteins, may be it could be degraded. I have proteins from September 2022.

Mohamed Khashan

Literaly this is what i get after scan my membrane

Dear Gareth Rostro

we are using the same stuff in our lab. This problem is related to the software settings, so it is better to contact the firm that supplied the instrument to get the problem solved.

best....

Mohamed Khashan Thankyou so much. I really apreciate your shares.

Regards

Gareth Rostro All the best

Hi everyone!

Thanks so much for your help.

Finally I could resolve the problem.

We use primary antibody 1:1,000 diluted in milk 1%, TBS-T 0.1% and secondary antibody 1:10,000 diluted in milk 1%, TBS-T 0.1%.

However the principal mistake was in our substrate, we change it and we got this.