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
- - - - -

Need help with my SDS-PAGE gels


  • Please log in to reply
9 replies to this topic

#1 Doraid

Doraid

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 22 September 2017 - 07:41 PM

Hello. 

I cast my gels using the BioRad recipe. I wash the wells with water multiple times before loading my samples. Yet, I always get these smear like lines. 

Are those due to the sample collection being less than optimal or the wells need better cleaning?

Attached Thumbnails

  • IMG_3892.JPG

Edited by Doraid, 22 September 2017 - 07:42 PM.


#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 24 September 2017 - 11:11 AM

it could be due to "skins" in the wells. they can form if the plates are warped or the spacers and comb don't match exactly (the comb may wear).

 

however, based on lanes 1 and 6, i suspect that the problem is the samples themselves (unless there is no sample in 1 and 6). is there kcl in the sample? it looks like a precipitate formed.


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

#3 Doraid

Doraid

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 25 September 2017 - 11:31 AM

it could be due to "skins" in the wells. they can form if the plates are warped or the spacers and comb don't match exactly (the comb may wear).
 
however, based on lanes 1 and 6, i suspect that the problem is the samples themselves (unless there is no sample in 1 and 6). is there kcl in the sample? it looks like a precipitate formed.

Lanes 1 and 6 are ladder lanes.
I use lysis buffer for collection and sonicate theb spin for an hour at 14K xg.

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 25 September 2017 - 12:23 PM

what's in your lysis buffer?

 

detergents like triton can displace sds and distort the run.


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

#5 Doraid

Doraid

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 25 September 2017 - 04:21 PM

what's in your lysis buffer?

 

detergents like triton can displace sds and distort the run.

150 mM sodium chloride 0.1% NP-40 (Triton X-100 can be substituted for NP-40) 50 mM Tris pH 8.0.

The way I do it, I use 200uL with 0.1% NP-40, sonicate then add 300uL lysis buffer without NP-40 and sonicate again. All have Phosphatase and protease inhibitors and DTT


Edited by Doraid, 25 September 2017 - 04:22 PM.


#6 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 26 September 2017 - 03:51 AM

both np-40 and triton x-100 can displace sds. also, the nacl can also distort the run.


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

#7 Doraid

Doraid

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 26 September 2017 - 10:13 AM

Okay. How can I get around this?

#8 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 26 September 2017 - 02:57 PM

you can do a buffer exchange to reduce salt and eliminate detergents in the sample prior to adding sds sample buffer. you an use spot dialysis if you want to prepare just enough for the gel sample.

 

you can dilute the sample with salt and detergent-free buffer if the protein is concentrated enough.


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

#9 Doraid

Doraid

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 26 September 2017 - 06:15 PM

Do you mind linking me to protocols to do do any of the suggested steps?

#10 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 27 September 2017 - 02:25 PM

for spot dialysis, float a piece of dialysis membrane on the buffer. then put a drop of sample on the membrane. no agitation.


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.