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.


Getting a SDS-page like picture in a PVDF blot??!!

Getting a SDS-page like picture in a PVDF blot??!! - Western Blot Forum

Getting a SDS-page like picture in a PVDF blot??!! - 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 07-08-2010, 04:01 AM
Pipette Filler
Points: 51, Level: 1 Points: 51, Level: 1 Points: 51, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Default Getting a SDS-page like picture in a PVDF blot??!!



Hi everyone,

I am new in WB and hope to get some helps from you guys in optimizing my runs. Thanks.

I am getting a SDS-page like picture in a PVDF blot, which is very unusual. Please have a look of the attached files. Do you have any idea on what went wrong with the runs? The ladder is a prestain ladder so it shouldn't be detected by a chemiluminescene system but I got it in my gel picture which is weird to me. I have tried to increase the washing time and tween buffer concentration in the wash buffer in order to reduce the background signal but it doesn't really work.

Following is the information of the runs.
1. Membrane used = PVDF membrane (pretreat with methanol for a few seconds before soak it in transfer buffer)
2. Detection system used = GAM HRP Detection System
3. Primary Antibody Dilution: 1:2,500
4. Secondary Antibodry Dilution 1:10,000
5. Expected protein size: 92 kDa
6. Transfer method: Semi-dry transfer with 48mA for 1 hr 45 mins.

Thanks again.

Best wishes,
Lee Yean
Attached Images
File Type: jpg 2010-07-07_12hr_58min-2.jpg (11.4 KB, 21 views)
Reply With Quote
  #2  
Old 07-08-2010, 02:02 PM
pigfarmer's Avatar
Summer Student
Points: 3,196, Level: 36 Points: 3,196, Level: 36 Points: 3,196, Level: 36
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2009
Location: Indiana
Posts: 99
Thanks: 26
Thanked 14 Times in 14 Posts
Default Re: Getting a SDS-page like picture in a PVDF blot??!!

I often see Ladder bands in my westerns as well. I think it may have something to do with leaving too much of the chemiluminescent on the membrane...not really a washing problem. Also, longer exposure times seem to give this result as well.

I do not think it is a problem though, if you look at the 'bands' they are really absense of background luminescence, not bands at all.
Reply With Quote
  #3  
Old 07-08-2010, 10:37 PM
Pipette Filler
Points: 1,174, Level: 19 Points: 1,174, Level: 19 Points: 1,174, Level: 19
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 18
Thanks: 0
Thanked 4 Times in 4 Posts
Default Re: Getting a SDS-page like picture in a PVDF blot??!!

Hi Lee Yean,

What kind of blocking condition you are using?

I suggest you try to use different kind of blocking condition, also, try to dilute your primary antibody to a lower concentration....

Good luck.
Reply With Quote
  #4  
Old 07-09-2010, 01:07 AM
Pipette Filler
Points: 51, Level: 1 Points: 51, Level: 1 Points: 51, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Getting a SDS-page like picture in a PVDF blot??!!

Hi Pigfarmer,
Thanks for your comment. But the bands were detected within the range of my expected protein size. So, I think there must be something I did wrong to get this reverse image (negative staining result). I'll decrease my exposure time on my next run, thanks :-)

Hi Pippuri,
I blocked my membrane after transfered using 3% BSA for 50mins at Room temperature. My supervisor also recommended me to use 5% skim milk powder, think I'll give it a try. thanks.

yeah, I need lots of luck now ... :-)
Reply With Quote
  #5  
Old 07-15-2010, 04:12 AM
Pipette Filler
Points: 51, Level: 1 Points: 51, Level: 1 Points: 51, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Getting a SDS-page like picture in a PVDF blot??!!

Hi All,

I got the bands I wanted after changing my blocking reagent from 3% BSA to 5% skim milk. The explanation from my assoc. supervisor was as follow. FYI.

" Looking at the result the most likely reason I can think is not blocking your membrane. If for instance by mistake you used buffer not containing the blocking protein, then in the next step your primary antibody would non-specifically bind to available sites all over the membrane and when you come in with the secondary antibody it will bind on the primary and any free sites still available thus you will get the dark background. The only places you will not get reaction is where your proteins already occupy the sites thus the negative staining."

cheers,
Lee Yean
Reply With Quote
Reply

Tags
blot , picture , pvdf , sdspage


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
dot blot and western blot Liang Western Blot Forum 1 08-07-2009 03:26 PM
Faint bands on PVDF blot. nsn SDS-PAGE Gel Electrophoresis Forum 1 04-09-2008 02:12 PM
protein flowthough in PVDF western Jayakumar, R Protocols and Methods Forum 1 09-05-2003 05:05 PM
FS: Old Physics-Related Books Oldbooks78 Physics Forum 0 08-27-2003 02:32 PM


All times are GMT. The time now is 05:39 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14096 seconds with 17 queries