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.


Transfer Issues to PVDF

Transfer Issues to PVDF - Western Blot Forum

Transfer Issues to PVDF - 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
  #11  
Old 10-01-2008, 11:15 PM
Pipette Filler
Points: 285, Level: 5 Points: 285, Level: 5 Points: 285, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2008
Posts: 6
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Transfer Issues to PVDF

Quote:
Originally Posted by danfive View Post
Try borrowing a power source or asking anyone about limiting the Amps.

If you are staining only one hour I suggest Imperial Protein Stain from Pierce, not the classic coomassie blue (that's usually an overnight stain with an overnight destain).

I hope you already know the amount of protein you are loading to each well, and perhaps a logical explanation to your different staining patterns is that the transfer gel had its excess SDS washed off (through the procedure) and thus stained more efficiently (that is if you also stained 1 hour etc).
hi danfive

You are absolutely right. The SDS was washed off by the methanol during the transfer, so I guess the gel wasn't washed enough. I am pretty sure SOME protein transferred over, but I just ordered some Ponceau S solution to stain the membrane and see if transfer occurred.

I'll let you guys know how it goes. As far as the actual western goes, using these films produce a glowing membrane and dark patch on the film, so I'm going to have to dilute the secondary antibody by 100-1000 I guess.
Reply With Quote
  #12  
Old 10-03-2008, 07:20 PM
Pipette Filler
Points: 285, Level: 5 Points: 285, Level: 5 Points: 285, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2008
Posts: 6
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Transfer Issues to PVDF

Good news guys.

I bought some ponceau s and stained my membrane after transfer at 15 V for 45 minutes.

BANDS WERE THERE YES!!!!!

Thanks for all your help guys.

Next step is to get this WB working .... haha,. Best of luck to all your experiments.

FOR ANYONE WHO HAS THE SAME PROBLEM:

- as people have told me in this thread, because protein is still in the gel doesn't mean that NO protein transferred.

- check membrane with some reversible stain if you're not sure

- once you're sure, just keep the same run time/volt etc.

Reply With Quote
  #13  
Old 04-07-2009, 07:32 AM
Pipette Filler
Points: 617, Level: 12 Points: 617, Level: 12 Points: 617, Level: 12
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2008
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Transfer Issues to PVDF

Hi guys,
our lab used to transfer at 0.8mA/cm^2 for 2 hours, Most importantly we keep 4 Volts at first .for example ,i have a 4*7.5 cm^2 gel ,i will transfer at 24 mA for hours and keep 4 volts.
Reply With Quote
Reply

Tags
issues , pvdf , transfer


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
New Saccharomyces Sequences 12/11/04 SGD Sequences Yeast Forum 0 12-12-2004 11:29 PM
New Saccharomyces Sequences 09/08/04 SGD Sequences Yeast Forum 0 09-13-2004 10:07 PM
protein flowthough in PVDF western Jayakumar, R Protocols and Methods Forum 1 09-05-2003 05:05 PM
uneven transfer of western blot- solved but.... Jayakumar, R Protocols and Methods Forum 0 08-28-2003 02:42 PM
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 09:49 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 0.12953 seconds with 16 queries