low and weak bands

[Andre212]

Hi everyone¡¡¡

Im new in this forum, and I would like to ask you one thing:

When I did a Western; b actin can see perfectly , but my protein see very bad. Firstly, I have tried to block less time(1 hour), but the results was background, then I tried to block more time(2 hours) but th results was many band(background).
I use 40 mg of protein.

What can I do?

Thank you very much.

[admin]

Hi Andre, welcome to the site. Can you let us know the method with steps you used. Did you blot for Actin first and then strip? Or the other way around.

cheers

[Andre212]

In my western, Im looking for portein HIF(120 KDa)

- Lower or resolving gel, 10 %.
- Uper or stackin gel, 4%.

Protein + "LSB5X with mercaptoetanol" (1:4) and 95ªC for five minutes.

The gel is running for 1 h, 150 V.
Transfer to the membrane PVDF, before membrane for five minutes in methanol, and then fiberpad, filter, membrane and gel in trans buffer for 5-10 minutes; 2h 200mA.

- Block with PBT(PBS +Tween)-BSA 5%, 1-3 hours, RT(I tried with non fat milk, but It didnt).
- Ab1ª, 1/1000, overnight, 4ºC.
-4xwash PBT 5 minute.
- Ab2ª, 1:5000, 1 hour, RT.
- 4xwash PBT 5 minute.
-ECL.

I have tired block more or less time, moreover I have tried more washing time.

[admin]

Hi Andre, I would try blocking during your secondary antibody incubation. Also have you tried to optimize your antibody conditions? you can cut pieces of transfer and blot each separately like:
1) 1/500 primary and 1/10000 secondary 2) 1/1000 primary and 1/10000 secondary etc.

Also you can do the same and try different blocking conditions, I would also suggest to use a positive control and check protein concentration/quality.

Can you post a picture of your gel?
good luck
cheers