Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Molecular Biology Techniques > Electrophoretic Mobility Shift Assay Forum
Register Search Today's Posts Mark Forums Read

Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


EMSA troubles

EMSA troubles - Electrophoretic Mobility Shift Assay Forum

EMSA troubles - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-18-2012, 10:51 PM
Pipette Filler
Points: 36, Level: 1 Points: 36, Level: 1 Points: 36, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2012
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default EMSA troubles



Hi everybody

i am doing the EMSA assay which made me mad, i used the Cy5 labeled primers to amplify the fragment for probe,PCR product was run on the agarose to confirm the unique PCR product. 1ul PCR product(with out gel purification) was used for EMSA, i always got additional band above the free probe, when i added less 0.5 ul PCR for EMSA, this additional band was less as well, do how can i address this problem? is it nessary to purify the PCR product before EMSA?

PS: lane1 and 4: only probe without protein
lande2 and 5: 0.025ug protein + 1ul and 0.5ul probe respectively
lane3 and 6 : 1ug protein + 1ul and 0.5ul probe respectively

waiting for your nice suggestions!

Best wishes,
dodo
Attached Images
File Type: jpg EMSA.JPG (33.9 KB, 32 views)
Reply With Quote
  #2  
Old 02-19-2012, 09:27 AM
admin's Avatar
Administrator
 
Join Date: Nov 2005
Posts: 1,418
Thanks: 883
Thanked 68 Times in 58 Posts
Default Re: EMSA troubles



Hi there, seems you are getting some streaking and gel issues. What running temperature and buffer (TAE vs TBE) are you using? What current/voltage?

Also seems you have a lot of signal, how much probe are you running on there?
Reply With Quote
  #3  
Old 02-19-2012, 10:09 PM
Pipette Filler
Points: 36, Level: 1 Points: 36, Level: 1 Points: 36, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2012
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: EMSA troubles

Hi there, seems you are getting some streaking and gel issues. What running temperature and buffer (TAE vs TBE) are you using? What current/voltage?

Also seems you have a lot of signal, how much probe are you running on there?[/quote]



Hi ,Thanks for your reply, i used the 0.5 TBE as running buffer at cold room 4 degree, and set the voltage at 120V, i Used 1ul and 0.5ul PCR products(50ul volume reaction ) for EMSA, but i did not purify the PCR products.

Xiaorong
Reply With Quote
  #4  
Old 02-21-2012, 10:19 PM
Banned
Points: 157, Level: 3 Points: 157, Level: 3 Points: 157, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2012
Posts: 46
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: EMSA troubles

thanks for u
Reply With Quote
Reply

Tags
emsa , troubles


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
Rna Emsa tara29466 RNA Techniques Forum 4 06-29-2011 10:35 AM
RNA EMSA Labeling Method with Biotin admin RNA Techniques Forum 5 10-12-2010 01:06 PM
EMSA with Proteins which bind non-specifically to DNA Adelaide_Science Electrophoretic Mobility Shift Assay Forum 2 12-10-2008 02:09 PM
EMSA problem Balat Protocols and Methods Forum 1 11-20-2006 08:39 AM
EMSA help...please URGENT sumo1 Protocols and Methods Forum 5 09-14-2006 09:50 AM


All times are GMT. The time now is 04:06 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 0.12710 seconds with 17 queries