Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biology Forum
Register Search Today's Posts Mark Forums Read

Biology Forum Biology Forums. Ask questions and discuss the study of Biology. If you have Biology questions from your homework ask them here!


Fusion protein

Fusion protein - Biology Forum

Fusion protein - Biology Forums. Ask questions and discuss the study of Biology. If you have Biology questions from your homework ask them here!


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 12-24-2010, 06:18 PM
Pipette Filler
Points: 1,078, Level: 18 Points: 1,078, Level: 18 Points: 1,078, Level: 18
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2009
Posts: 8
Thanks: 0
Thanked 0 Times in 0 Posts
Default Fusion protein



Hi,i fused 2 genes,cloned and expressed in BL21.1 gene of 62 KDa and other gene of 60 KDa (totally fusion protein of 122 KDa).I checked with one antibody(62 KDa) in western blot,surprisingly instead of 122KDa band size ,I got only 62 KDa band size!!! Even if i use one antibody,my blot sud come in 122 KDa is it? Say me why??
After cloning my fusion gene I digested and checked.Cloning is ok.
Reply With Quote
  #2  
Old 10-14-2011, 04:30 PM
Pipette Filler
Points: 24, Level: 1 Points: 24, Level: 1 Points: 24, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2011
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Fusion protein

did u double check if the stop codon has been removed from the first gene? if it is still there, then of course only the first gene will be translated. hope it helps
Reply With Quote
  #3  
Old 10-15-2011, 07:04 PM
Graduate Student
Points: 1,231, Level: 20 Points: 1,231, Level: 20 Points: 1,231, Level: 20
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2011
Posts: 113
Thanks: 0
Thanked 16 Times in 13 Posts
Default Re: Fusion protein

Quote:
Originally Posted by Chitra View Post
Hi,i fused 2 genes,cloned and expressed in BL21.1 gene of 62 KDa and other gene of 60 KDa (totally fusion protein of 122 KDa).I checked with one antibody(62 KDa) in western blot,surprisingly instead of 122KDa band size ,I got only 62 KDa band size!!! Even if i use one antibody,my blot sud come in 122 KDa is it? Say me why??
After cloning my fusion gene I digested and checked.Cloning is ok.
The protein could be getting cleaved in half in the bacteria. Proteolysis can be a real problem when expressing eukaryotic proteins in bugs. Is there some kind of linker peptide between the two genes? perhaps try modifying the linker.

I had the problem that my protein has a domain at its N-terminus and a domain at its C-terminus, but the middle is predicted to be largely unstructured. When expressed in bacteria the protein gets chopped in half into the two stable domains. That might be what happens to your protein.

If you leave your blot down for a really long time you might be able to see a faint band at 122 kDa.
Reply With Quote
  #4  
Old 10-19-2011, 05:11 PM
Warthaug's Avatar
Post-Doc
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: Fusion protein

A few ideas:
1) Check your sequence; make sure everything is in-frame and that you removed the 1st stop codon.
2) mmorgan's point is a good one; bacterial protease inhibitors are available; try culturing a short while with those and then check for your protein via western.
3) If you have an Ab against the 2nd gene try doing a western with that; it'll let you know if the whole protein is being expressed (verses only the first half being expressed).
4) Try another expression system - insect, yeast or mammalian cells for example.

Bryan
Reply With Quote
  #5  
Old 11-10-2011, 08:55 PM
Pipette Filler
Points: 72, Level: 1 Points: 72, Level: 1 Points: 72, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Posts: 6
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Fusion protein

it has really had an impact on me - at long last I am actually starting to understand it! I understood your writing! *having a eureka moment here!* Thank you
Reply With Quote
  #6  
Old 11-10-2011, 08:56 PM
Pipette Filler
Points: 72, Level: 1 Points: 72, Level: 1 Points: 72, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Posts: 6
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Fusion protein

I don't believe on niche because I thinks that If someone feels that he should own a blog then he should thinks that his blog will contain almost all the topics and people from all age will feel happy to browse your blog.
Reply With Quote
  #7  
Old 11-10-2011, 08:57 PM
Pipette Filler
Points: 72, Level: 1 Points: 72, Level: 1 Points: 72, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Posts: 6
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Fusion protein

This post is very interesting, I like it. I will always come to visit after.I would recommend to friends more.
Reply With Quote
  #8  
Old 11-22-2011, 08:52 AM
Aisha Bennett's Avatar
Pipette Filler
Points: 22, Level: 1 Points: 22, Level: 1 Points: 22, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Fusion protein

Just check whether the fusion protein is continuous with the target protein or not. You need to maintain the same open reading frame. And most importantly omit the stop codons between the target protein and the fusion partner.
Reply With Quote
Reply

Tags
fusion , protein


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 - 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
New Saccharomyces Sequences 09/10/03 SGD Sequences Yeast Forum 0 09-11-2003 01:33 AM


All times are GMT. The time now is 10:15 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 0.20768 seconds with 16 queries