Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Prey protein band bleeds, IgG band normal?


  • Please log in to reply
3 replies to this topic

#1 chelsea77

chelsea77

    member

  • Members
  • Pip
  • 2 posts
1
Neutral

Posted 28 August 2013 - 08:06 AM

Very frequently, when I run my Co-IP samples, the prey protein band will bleed into adjacent lanes.  Conversely, the input for these proteins retains good lane discipline and the bands are clearly separated.

 

Moreover, the protein I pull down does not experience this issue, and the heavy/light chain bands also resolve quite nicely into separate lanes.  As a corollary, the pulled-down protein has a similar MW to the prey proteins (e.g., within ~15kD):  if there was a problem with the gel, then the IgG should look funny too, and most definitely the pulled-down protein.

 

Thanks for any help you can provide.


Edited by chelsea77, 28 August 2013 - 08:14 AM.


#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,735 posts
125
Excellent

Posted 03 October 2013 - 10:30 AM

the only things i can think of are:

 

too much protein loaded

 

additives in the co-ip buffer (eg salts, detergents)

 

pH of the co-ip buffer (its effect on the sds sample buffer)


talent does what it can
genius does what it must
i do what i get paid to do

#3 chelsea77

chelsea77

    member

  • Members
  • Pip
  • 2 posts
1
Neutral

Posted 07 October 2013 - 09:11 AM

mdfenko,

Thanks for the ideas.  My guesses are between too much protein loaded and pH of the co-IP buffer.  Specifically, I am worried that the beads may affect the pH.  Very frequently, after I boil my samples in sds sample buffer, the liquid in IP/beads tubes change color from blue to green (and, in extreme cases, yellow).  However, the lysate/input tubes stay blue.  Next time I do IP I will use a newer aliquot of Tris (my Flag lysis buffer calls for 50mM so I probably can't use any more). 

 

Thanks again!!!



#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,735 posts
125
Excellent

Posted 08 October 2013 - 11:23 AM

if the tracking dye is turning green to yellow then the pH is too low. you can add a few ul (or less) of 0.5M (or lower) tris base (or pH 9) to the sample to raise the pH until the dye turns blue.

 

the lower pH does have an effect on migration.


talent does what it can
genius does what it must
i do what i get paid to do




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.