Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Western Blot Forum
Register Search Today's Posts Mark Forums Read

Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Problem with Western Blot Transfer Conditions?

Problem with Western Blot Transfer Conditions? - Western Blot Forum

Problem with Western Blot Transfer Conditions? - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 04-29-2009, 10:47 AM
Pipette Filler
Points: 94, Level: 1 Points: 94, Level: 1 Points: 94, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Question Problem with Western Blot Transfer Conditions?



Hello for everyone!
I'm new on this Forum.
I'm using PVDF membrane, and I'm doing the right treatment, whit methanol (30 seconds), water MilliQ (1 minute) and transfer buffer (10-15 minutes).
When I do the Ponceau S test after the transfer, the bands don't show up!!!
I'm think I'm having a problem whit the transfer!!
Here's my conditions to transfer the proteins:
- For 1 gel: 100V and 200 mA for 2 hours at cold room. But after 30 minutes, the V falling down to 40-50 V.
-For 2 gels: 140 V and 400 mA for 2 hours at cold room. After 30 minutes the V falling down to 65-70 V.

What is my problem? Is the V???
What do you suggest me? transfer overnight whit a low V (30V)?
Thanks for help me
Reply With Quote
  #2  
Old 05-28-2009, 11:53 PM
admin's Avatar
Administrator
 
Join Date: Nov 2005
Posts: 1,418
Thanks: 883
Thanked 68 Times in 58 Posts
Default Re: Problem with Western Blot Transfer Conditions?

Hi Renno,
have you tried a fresh newly made transfer buffer? Seems that may be the issue or your transfer apparatus.

I would transfer overnight at 30V also, much better as you are transferring at high voltage but you mention it falls to 40-70V which is not enough after 30 minutes for a good transfer. Seems your buffer is having issues.
Reply With Quote
  #3  
Old 05-29-2009, 11:50 AM
Anastasia_S's Avatar
Summer Student
Points: 326, Level: 6 Points: 326, Level: 6 Points: 326, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2009
Posts: 70
Thanks: 3
Thanked 12 Times in 12 Posts
Default Re: Problem with Western Blot Transfer Conditions?

I transfer at 70V for 12 h. Now you made me think I'm overdoing it!
Reply With Quote
  #4  
Old 05-30-2009, 11:19 PM
Pipette Filler
Points: 94, Level: 1 Points: 94, Level: 1 Points: 94, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Problem with Western Blot Transfer Conditions?

Hello again, and thanks for answer me.

Well, I chance some products of my transfer buffer, and the way that I make the "sandwich step", and now the thinks are ok again!!!
I think the problem was in the transfer step! Thank you Admin.

Admin, to transfer 30v overnight, what mA should I need to use?

Anastasia_S, my transfer stills 60 V (55-60v), 200 mA (for 1 gel) for 2 hours on ice, and I think it's enough time to transfer. I didn't have problem anymore.
Maybe for protein whit high molecular weight, you'll need more time. I can transfer in 2 hours, proteins with 70-72 kda for example.

See you soon!!!

Renno
Reply With Quote
Reply

Tags
blot , conditions , problem , transfer , western


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Can we use dot blot inplace of Western blot? peterish Western Blot Forum 3 06-16-2010 07:59 PM
Cloudy Western Blot wexfrdo Western Blot Forum 1 08-26-2008 10:18 PM
Western problem Jayakumar, R Cell Biology and Cell Culture 0 12-31-2003 02:25 PM
uneven transfer of western blot Wolfgang Schechinger Protocols and Methods Forum 0 08-23-2003 10:47 AM
uneven transfer of western blot EK Protocols and Methods Forum 0 08-21-2003 05:36 PM


All times are GMT. The time now is 02:44 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12996 seconds with 16 queries