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
- - - - -

problem of 2D-GE with phagosome fractions


  • Please log in to reply
7 replies to this topic

#1 Myco Rax

Myco Rax

    member

  • Active Members
  • Pip
  • 19 posts
1
Neutral

Posted 17 April 2011 - 08:16 PM

Hi,
I prepare mycobacaterial phagosomes from the MDM (monocyte derived macrophages).
AFter which i need to perform 2D-GE. I tried based on the available literature, but unfortunately none gives proper complete description of sample preparation and also program for isoelectric focussing. can anyone plz give a proper protocol regarding the sample preparation and also IEF program.
Thanks

#2 proteaMatt

proteaMatt

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
4
Neutral

Posted 18 April 2011 - 05:26 AM

Could we see what you have tried so far? That would help people troubleshoot the problems you are having.
Lab Technician at Protea Biosciences

#3 Myco Rax

Myco Rax

    member

  • Active Members
  • Pip
  • 19 posts
1
Neutral

Posted 20 April 2011 - 01:41 AM

Could we see what you have tried so far? That would help people troubleshoot the problems you are having.

Purified phagosome fractions using sucrose density gradients (Salt (EDTA, EGTA, Imidazole) concentrations are lesser than 20mM (Combined) in homogenization buffer).
I boiled my phagosome fractions in 40mM Tris pH8, 0.25% SDS and 100mM DTT. then dried the samples using speedvac. I then use a lysis solution of 9.9M urea and 4% CHAPS (to dilute out SDS), 2% ampholytes 3-10. Passive rehydration (3-10NL strips). then focussing using the protocol 0-250V (1 hr), 250V (1.5 hr), 250-3000V (4 hr), 3000v (15000vhr). the run 12% SDS-PAGE. I tried varyin the Tris (10mM, 20mM, 30mM) concentrations, as i cud horizontal streaking in the gel. Also tried reduced SDS (0.2%) concentration.
but my gel still shows horizontal streaking and the spots are more concentrated towards acidic end.
Please help me trouble shoot it.
Thanks in advance

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,742 posts
127
Excellent

Posted 20 April 2011 - 08:07 AM

you may not have completely dissociated the sds from the proteins. you can try adding triton x-100 instead of or with the chaps.
talent does what it can
genius does what it must
i do what i get paid to do

#5 Myco Rax

Myco Rax

    member

  • Active Members
  • Pip
  • 19 posts
1
Neutral

Posted 20 April 2011 - 08:16 AM

you may not have completely dissociated the sds from the proteins. you can try adding triton x-100 instead of or with the chaps.


Well, Can you please suggest the conc. of triton X-100?

#6 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,742 posts
127
Excellent

Posted 20 April 2011 - 08:24 AM

you can start with 1% and go from there (when dialyzing you use equal concentration, the sds will be diluted by the dialysis solution).

are you sure your urea is intact. many people warm up their solution to completely dissolve urea quickly but that will actually cause the urea to decompose. the solution should be made at temperatures no higher than room temperature. it will take longer but it will be urea.
talent does what it can
genius does what it must
i do what i get paid to do

#7 Myco Rax

Myco Rax

    member

  • Active Members
  • Pip
  • 19 posts
1
Neutral

Posted 21 April 2011 - 01:26 AM

you can start with 1% and go from there (when dialyzing you use equal concentration, the sds will be diluted by the dialysis solution).

are you sure your urea is intact. many people warm up their solution to completely dissolve urea quickly but that will actually cause the urea to decompose. the solution should be made at temperatures no higher than room temperature. it will take longer but it will be urea.


I am sure that urea i use is intact. I never dissolve it by warming the solution.
I'll try with triton x-100. thanks for that.
Any other suggestions?

#8 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,742 posts
127
Excellent

Posted 21 April 2011 - 10:54 AM

normally you would (at least partially) defeat streaking in ief by running for a longer period of time (with immobilized pH gradients you won't experience significant cathode drift).

since your samples are drifting toward the acid side (probably caused by the presence of sds) you can't extend the run.

after you optimize the triton then you can extend the run, if necessary.
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.