Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Problems of western signal

western

  • Please log in to reply
5 replies to this topic

#1 cell 293

cell 293

    member

  • Active Members
  • Pip
  • 17 posts
1
Neutral

Posted 01 September 2014 - 09:11 PM

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.

 

1.jpg

 

 

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

 

2.jpg  

 

 

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?

 

3.jpg

 

 

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. 


Edited by cell 293, 01 September 2014 - 09:11 PM.


#2 mdfenko

mdfenko

    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,327 posts
212
Excellent

Posted 02 September 2014 - 04:21 AM

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).


talent does what it can
genius does what it must
i used to do what i got paid to do


#3 cell 293

cell 293

    member

  • Active Members
  • Pip
  • 17 posts
1
Neutral

Posted 02 September 2014 - 07:32 AM

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?



#4 mdfenko

mdfenko

    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,327 posts
212
Excellent

Posted 03 September 2014 - 03:29 AM


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.


talent does what it can
genius does what it must
i used to do what i got paid to do


#5 cell 293

cell 293

    member

  • Active Members
  • Pip
  • 17 posts
1
Neutral

Posted 03 September 2014 - 07:20 AM

Thanks your reply

 

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



#6 mdfenko

mdfenko

    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,327 posts
212
Excellent

Posted 03 September 2014 - 08:17 AM

 

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.


talent does what it can
genius does what it must
i used to do what i got paid to do






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.