Problems of western signal - (Sep/02/2014 )

Hi everyone

 

I have problem to improve my weak signal.

 

I expect to gel the signal at 35, 50, and 103 kDa. In my data, i have to expose and get the signal around 10~15 min. But the signal is still unclear.

 

What can i do to improve it.

 

In this data, background is every high, but the signals what i want are weak.

 

 

 

In this data. How to get stronger signal? And how to removal the black point in the film?

 

 

 

 

This is beta-actin. Sometimes i got two signals when numerous sample was loaded. I am sure i didn't move the film when it was exposed.

 

Is it normal? How to imporve it?

 

 

 

This is simple protocol of my western.

 

1. Dissolve cells by adding PBS and 2X sample buffer  and boiled directly.   (I don't lysis because protein will be lose.)

 

2. Start the electrophoresis process for the sample at 60 V.

 

3. Thansfer proteins at 300 mA, 2 hr.

 

4. Block membrane with 5% non-fat milk in TBS

 

5. Incubation with primary antibody (goat, 1:500) at 4℃ overnight (around 16 hr)

 

6. Wash 10 min with TBST (tween 20 0.1%), 3 times.

 

7. Incubation with secondary antibody (1:5000) at room temperature 1 hr.

 

8.  Wash 10 min with TBST (tween 20 0.1%), 3 times.

 

9. ECL

 

Thanks a lot. 

how does a stained gel look?

 

have you ponceau stained the membrane to see how well the transfer went?

 

the first blot looks like there are transfer artifacts (pattern from the filter paper or electrode (if semi-dry) and/or uneven distribution of solutions during incubations (could be caused by the membrane landing on the bottom of the vessel and sticking).

 

you may be able to improve the strength of signal by adjusting the antibody concentrations (you can reduce some background by including some blocking agent in the antibody solution).

Thanks for your reply.

 

I have  ponceau stained the membrane. It is no problem to transfer.

 

Do you mean to dilute primary antibody with non-fat milk in TBS?

 

Is it work to adjust the secondary antibody concentration?

cell 293 on Tue Sep 2 15:32:14 2014 said:

Do you mean to dilute primary antibody with non-fat milk in TBS?

 

Is it work to adjust the secondary antibody concentration?

dilute with nfm in tbst.

 

i would try adjusting the primary antibody to increase signal before adjusting the secondary. has the secondary been used successfully at that concentration? if so then it probably won't require adjustment.

 

you can determine if the background is being caused by the primary antibody by processing a parallel blot without primary antibody.

Thanks your reply

 

Do you mean i can prepare a membrane that is only with secondary primary? 

cell 293 on Wed Sep 3 15:20:24 2014 said:

 

Do you mean i can prepare a membrane that is only with secondary primary? 

yes. you do everything the same but omit the primary antibody step (and, of course, the following wash). this way you will find out if your secondary antibody is binding to anything on the membrane.