Hi Srichandrasekar Indhuprakash

Just a few general queries:

Did you get rid of the air bubbles?

Was your antibody mono- or polyclonal?

How many washes did you give in between?

Which blocking buffer did you use?

Varsha

Hi,

Could your issue be with ECL substrate and antibody dilution rather than protein transfer? I am assuming that you quantified the protein that you are running on the gel by some colorimetric analysis and are loading equal volumes in each well. I also do not know which type of transfer method you are using - wet, semi-dry, rapid? transfer efficiency varies a lot among these methods. I use the wet transfer method and let the transfer continue for 2:30 hours at 4 degrees. after transfer you can check by ponceau whether your transfer happened properly or not, blocking optimization is another issue, we usually block for 1 hour at room temperature. then primary antibody dilution and incubation time is also something which can be optimized, ECL substrate ratio (if you are using bio rad clarity and clarity max) also can be optimized from 1:10 to 1:1 ratio (clarity: clarity max). I would suggest that you run a positive control (any lysate which expresses your protein of interest in abundance) along with your gel to resolve the issues with antibody and blot development. I need more information on your process in order to pinpoint the issue but hope it helps.

Varshasnata Mohanty Hi,

1. There were no air bubbles in the membrane after the transfer.

2. My antibody is polyclonal

3. Three washes were given in each step

4. I used BSA for blocking

Vishal Sah Hi,

I have quantified my protein by the colorimetric method and loaded 100 micrograms of protein in each well.

I tried both wet and rapid transfer, I can able to see the protein bands clearly on the rapid transfer. The transfer time was 1.30hrs for wet transfer and 8 minutes for rapid one.

Blocking time is the same as you mentioned.

The ECL substrate was not the issue it's a newer one from the cyanogen brand.

Hi Srichandrasekar Indhuprakash

Thank you so much for the answers. The primary antibody might need to be optimally diluted. You can also try using skimmed milk (3-5%) for blocking. I mean these are just suggestions and the problem could be anywhere. As Vishal Sah has already pointed out, it could be ECL too! Please try the same procedure with any known control such as HeLa lysate and look for any housekeeping genes. It is just to rule out any technical issues.

Varsha

Varshasnata Mohanty

Thank you for the response and suggestions.

I will look into it.

IMHO, PVDF membranes for transfer and non-fat dry milk for blocking give better results than BSA and NC. I also find tank blotters more reliable than semi-dry, probably because the Ohmian heat is distributed over a larger volume. Dunn_s transfer buffer (DOI:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3, 50 µM SDS for larger proteins) works better than Towbins.

When you assemble the cassette, work in a tea tray or similar, and use plenty of transfer buffer so that no air bubbles are trapped.

After transfer, stain the blot with Ponceau Red (DOI:10.1016/0003-2697(86)90263-0) and the gel with CBB, to ensure that all protein has been transferred. Also stain a gel before transfer, if you are unsure that your protein sample and electrophoresis conditions are ok.

I mostly agree with Engelbert Buxbaum's answer and I will recommend this with some suggestions top of it. Firstly, if you are developed with X-ray film then use nitrocellulose membrane which give you better results because of high affinity for protein and high retention ( avoid the methanol) or if you are using SuperSignal west pico/femto substrate and your protein is hydrophobic then use PVDF membrane (with pre-soaking with methanol). Secondly, the wet transfer process can be done in 4 degree chamber/in a big ice filled ice bucket where ice should be covered around your buffer tank, use the blue cooling unit (should be in -20 degree prior to use) into buffer tank/cooling coil, also use chilled transfer buffer during assemble the cassette as well as in the buffer tank also.