Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Basic Lab Protocols and Techniques > SDS-PAGE Gel Electrophoresis Forum
Register Search Today's Posts Mark Forums Read

SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


Protein does not migrate into resolving gel

Protein does not migrate into resolving gel - SDS-PAGE Gel Electrophoresis Forum

Protein does not migrate into resolving gel - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 11-25-2011, 09:17 AM
Pipette Filler
Points: 44, Level: 1 Points: 44, Level: 1 Points: 44, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Protein does not migrate into resolving gel



I am currently trying to investigate an enzyme secreted from an unknown marine bacteria. I have collected supernatant from bacterial shake flask cultures and aim to analyze these supernatants via SDS-PAGE to identify the size of the enzyme and possibly use MS to sequence the enzyme.

Problem is that the protein is not migrating into the resolving gel. Both my protein marker (Fermentas Prest. Prot.) and the dye front of the sample buffer seems to migrate properly, but when the gel is stained with Coomassie, the only band I get is an extremely smeary one at the stacking/resolving gel interface. It seems like the sample has "stuck" at the resolving gel and then just diffused throughout the stacking gel.

I use BIO-RAD precast Mini Protean TGX gels of 4-20% and a MOPS running buffer. Running gels at 180V for ca 40 minutes. Samples are boiled prior to loading and the loading buffer contains appropriate amounts of BME.

Any ideas?
Reply With Quote
  #2  
Old 11-25-2011, 12:22 PM
obama's Avatar
Graduate Student
Points: 2,830, Level: 34 Points: 2,830, Level: 34 Points: 2,830, Level: 34
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Hannover Germany
Posts: 145
Thanks: 2
Thanked 25 Times in 21 Posts
Default Re: Protein does not migrate into resolving gel

Hi lindberd,

Add some more points like:

How you processed the supernatant: Which buffer you dissolved it, did you boiled it before loading. Did you measured the protein conecntration if yes then how much you loaded.

Then one can get an idea what's going wrong.
Reply With Quote
  #3  
Old 01-22-2013, 08:14 AM
Volunteer
Points: 155, Level: 3 Points: 155, Level: 3 Points: 155, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2013
Posts: 54
Thanks: 0
Thanked 0 Times in 0 Posts
Default アグ 激安

[

[

[
Reply With Quote
  #4  
Old 02-11-2013, 06:14 AM
Summer Student
Points: 260, Level: 5 Points: 260, Level: 5 Points: 260, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 54
Thanks: 0
Thanked 0 Times in 0 Posts
Default エアジョーダン 1

[

[

[
Reply With Quote
  #5  
Old 02-11-2013, 06:17 AM
Summer Student
Points: 260, Level: 5 Points: 260, Level: 5 Points: 260, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 54
Thanks: 0
Thanked 0 Times in 0 Posts
Default ジョーダンスニーカー

[

[

[
Reply With Quote
  #6  
Old 02-11-2013, 07:50 AM
Summer Student
Points: 260, Level: 5 Points: 260, Level: 5 Points: 260, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 54
Thanks: 0
Thanked 0 Times in 0 Posts
Default エアジョーダン 2013

[

[

[
Reply With Quote
  #7  
Old 02-11-2013, 07:58 AM
Summer Student
Points: 260, Level: 5 Points: 260, Level: 5 Points: 260, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 54
Thanks: 0
Thanked 0 Times in 0 Posts
Default ジョーダン 11

[

[

[
Reply With Quote
  #8  
Old 02-11-2013, 12:19 PM
Summer Student
Points: 246, Level: 4 Points: 246, Level: 4 Points: 246, Level: 4
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 95
Thanks: 0
Thanked 0 Times in 0 Posts
Default ベルスタッフ ジャケット

[

[

[
Reply With Quote
  #9  
Old 02-11-2013, 12:47 PM
Summer Student
Points: 246, Level: 4 Points: 246, Level: 4 Points: 246, Level: 4
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 95
Thanks: 0
Thanked 0 Times in 0 Posts
Default ベルスタッフ バッグ

[

[

[
Reply With Quote
  #10  
Old 02-11-2013, 12:52 PM
Summer Student
Points: 246, Level: 4 Points: 246, Level: 4 Points: 246, Level: 4
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 95
Thanks: 0
Thanked 0 Times in 0 Posts
Default ベルスタッフ アウトレット

[

[

[
Reply With Quote
Reply

Tags
gel , migrate , protein , resolving


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
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Human Cytome Project - an idea - Update 19 April 2005 Peter Van Osta Cell Biology and Cell Culture 1 06-01-2009 02:17 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM
New Saccharomyces Sequences 11/27/04 Mike Cherry Yeast Forum 0 11-29-2004 12:39 AM
New Saccharomyces Sequences 05/19/04 SGD Sequences Yeast Forum 0 05-23-2004 04:06 PM


All times are GMT. The time now is 05:48 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 0.21298 seconds with 16 queries