What is your current protocol for western blotting? There are lots of different possibilities.

1. Did your gel transfer to the membrane properly? Do you have ladder bands that you can see on the transfer?

-is the membrane good, the right type

-set up of apparatus (bubbles, etc)

-manufacturers recommendations for preparing membrane (i.e. some membranes require a short bath in alcohol before putting them in buffer)

2. Are you blocking? How long?

3. Did you try different dilutions of antibody? Are you using a primary and secondary, or just a labeled primary?

4. How are you detecting your blot? Change incubation time/exposure?

Good luck!

Hi

I know there are two ways for western blot detection systems

Alkaline phosphates and HRP . Ive tries both of them if thats what you mean by western blot protocol.if not please explain that for me .

I can see nothing in the membrane and gel because im using commassi blue and Ponceau S and both of them not senstive enough to detect the very diluted protein like insulin in the milk . Thats way im doing the western blot .

Regading to the membrane im not sure how can i know if it is the right type im useing nitrocelluse membrane and ive tried the PVDF and i followed the company provider advices in working with both membran.got same result that ive got same result

Tranferring done perfectly no puples were there

And i used marker as well

I blocked membrane with 5% BSA for 1 hour

I used primary and secondary and i used positive control (human insulin)

Incubation time my supervisor said it must not excess 30 min atherwice none specific binding can happen

I hope that im answering all questions . really need help in that

So you can detect the human insulin on your western blot (on the same blot as your camel sample) with no problem? If the transfer is fine, detection of human insulin is fine, etc etc then most likely your protein is just too dilute.

Even though no one likes radioactivity, you will get the best sensitivity with a radioactive antibody as opposed to alkaline phosphatase or HRP. If you can, I would try that. You can also expose radioactivity to film for longer as the signal keeps going longer.

I think your other option would be to concentrate your sample before putting it on a gel. I've never tried to concentrate something like camel's milk, but maybe you could use something like an amicon ultra?

I've usually blocked with milk, but maybe that would interfere with your system (since you're looking for something that might be in milk?).

Also can you somehow add western blot to your title? I think you might get more answers. :)