That the anti-actin WB works fine tells you that the procedure and reagents are OK and the problem lies in the antibodies. You have to determine the optimum dilution for the anti-Egr1 antibody; 1:1000 might be too concentrated (I mean, even the size standards light up in your membrane). Also, perhaps the secondary antibody is too concentrated; you have to try lower concentrations there as well.

Hope it helps!

That the anti-actin WB works fine tells you that the procedure and reagents are OK and the problem lies in the antibodies. You have to determine the optimum dilution for the anti-Egr1 antibody; 1:1000 might be too concentrated (I mean, even the size standards light up in your membrane). Also, perhaps the secondary antibody is too concentrated; you have to try lower concentrations there as well.

Hope it helps!

I agree with Alejandro Martin. You can first of all change your milk concentration from 5% to 2.5% or even 1% (for most antibodies 1% milk should do), and for my antibodies I usually go with 1:2000 for the primary antibody and 1:10000 for the secondary antibody.

also for the blocking, we incubate our membrane for only 30mins in milk, maybe reduce the time a little bit more.

there was also one other trick that I used to do (not for SDS gels but for my blue native gels) and it made me get better bands , and that was that I used to incubate my gels for 30min in SDS denaturing buffer before blocking in milk solution.

SDS denaturing solution:

3.3% (w/v) SDS

4% (v/v) mercapto-ethanol

65 mM Tris-HCl pH 6.8

(this technique was based on a paper from Shingo Kikuchi.

just keep in mind to make the Buffer mentioned freshly!

hope it helps.

Hi there,

The background being very strong you may try to add BSA in your blocking buffer. Adjusting primary and secondary antibody dilutions is the obvious issue. As you don't exhibit any positive control, the reactivity of both antibodies may also be questioned.

Hi,

Beta actin blot being so neat ( but u decrease the concentration of beta actin antibody and exposure time to lessen the intensity as bands are so thick).

Regarding your second blot, so many unspecific bands in the blot can be because of so many reasons, so you need to do few steps: 1) Increase the time of blocking (You didnot mention the time of blocking). 2) Can try blocking using 5% BSA if you are having problem with 5% Skim milk. 3) Run a positive control for your antibody 4) Optimise the antibody dilution ( 1:1000 and 1:2000) 5) Keep the secondary antibody dilution, as signals we see on the blot is due to secondary antibody. 6) Increase your washing with TBST after secondary antibody incubation.

I hope it will solve your issue.

Hi,

Other than what has already been recommended, you can increase washing to 10 min each (thrice). You can also try adding BSA blocking solution along with your diluted primary antibody.

Good luck!

Hi

In my opinion you should

1) Do Overnight blocking (at 4C)

2) Prolong the primary antibody incubation upto 16 hrs. ( I would assume that u r incubating it for 8-12 hrs)

3)Decrease the sample boiling time up . to 5 minutes.

4) See if your proteins are resolved on the gel itself in the first place by conventional CBB staining and destaining.

5)Decrease the sample run voltage to 50V (while in stacking gel) and up to 150V max (while in resolving gel).

6) Increase the transfer time up to 3 hrs or may be O/N. You can decrease the voltage accordingly

7)After all this you can go for different range of your pri antibody and at what dilution would it give u the best signal. In my opinion anything from 1:250 to 1:3000 should be enough. Make 3 or 4 antibody dilution in this range. By looking at your actin i think u should dilute it more. As mentioned by other researchers those bands are really too thick.

Hope it helps!!