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
- - - - -

Runing problems


  • Please log in to reply
18 replies to this topic

#1 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 24 October 2013 - 04:51 AM

Hi to everyone!
I really need your help. I´m starting in a new lab and I making some WB, which I made thousands of them during my predoc term but I´m getting some problems, This is the fifth time I made this WB and I´m getting crazy right now because I made new solution for everything.
 
Let me give you some inf: White adipose tissue samples, lysis buffer (Hepes 50mM, NaCl 200mM, Glycerol 5%, Triton X100 1%, Ortovanadate, Naf, Complete), 9% acrilamide, 25uL sample into 1mm BioRad Gels, around 20ug protein, runing at 100V.
 Please, see the pic and you will see a "white ghost band" in the middle of the gel, and I do not what is this because I am using the same protocol since 4 years ago, I just change the lab... (maybe some negative energy... hehehe, joke)
 
Please, please, and please, could you help me to resolve this problem? Have you ever seen something like this?
 
Thanks so much in advance!!!!
 
email: vemovied@gmail.com


Edited by Vemovied, 24 October 2013 - 05:24 AM.


#2 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 310 posts
29
Excellent

Posted 24 October 2013 - 08:46 AM

Hey Vemovied,

 

I don't see a pic.  Can you try to attach it again?



#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,616 posts
118
Excellent

Posted 25 October 2013 - 04:17 AM

were you using adipose tissue in the past?


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

#4 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 27 October 2013 - 04:50 AM

Hi to everyone!
I really need your help. I´m starting in a new lab and I making some WB, which I made thousands of them during my predoc term but I´m getting some problems, This is the fifth time I made this WB and I´m getting crazy right now because I made new solution for everything.
 
Let me give you some inf: White adipose tissue samples, lysis buffer (Hepes 50mM, NaCl 200mM, Glycerol 5%, Triton X100 1%, Ortovanadate, Naf, Complete), 9% acrilamide, 25uL sample into 1mm BioRad Gels, around 20ug protein, runing at 100V.
 Please, see the pic and you will see a "white ghost band" in the middle of the gel, and I do not what is this because I am using the same protocol since 4 years ago, I just change the lab... (maybe some negative energy... hehehe, joke)
 
Please, please, and please, could you help me to resolve this problem? Have you ever seen something like this?
 
Thanks so much in advance!!!!
 
email: vemovied@gmail.com

o9lb3s.jpg


Edited by Vemovied, 27 October 2013 - 08:14 AM.


#5 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 27 October 2013 - 08:13 AM

Hi to everyone!
I really need your help. I´m starting in a new lab and I making some WB, which I made thousands of them during my predoc term but I´m getting some problems, This is the fifth time I made this WB and I´m getting crazy right now because I made new solution for everything.
 
Let me give you some inf: White adipose tissue samples, lysis buffer (Hepes 50mM, NaCl 200mM, Glycerol 5%, Triton X100 1%, Ortovanadate, Naf, Complete), 9% acrilamide, 25uL sample into 1mm BioRad Gels, around 20ug protein, runing at 100V.
 Please, see the pic and you will see a "white ghost band" in the middle of the gel, and I do not what is this because I am using the same protocol since 4 years ago, I just change the lab... (maybe some negative energy... hehehe, joke)
 
Please, please, and please, could you help me to resolve this problem? Have you ever seen something like this?
 
Thanks so much in advance!!!!
 
email: vemovied@gmail.com



#6 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,616 posts
118
Excellent

Posted 28 October 2013 - 05:24 AM

the white band appears to be ahead of or with a buffer front. it could be solubilized lipid (most likely in micelles) that is migrating due to eof.


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

#7 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 28 October 2013 - 06:52 AM

the white band appears to be ahead of or with a buffer front. it could be solubilized lipid (most likely in micelles) that is migrating due to eof.

Thanks so much for your help, but it never happens to me and I am always using adipose tissue... Do you know how I can avoid this fat?



#8 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,268 posts
213
Excellent

Posted 28 October 2013 - 03:54 PM

You could extract the fat with a chloroform wash.



#9 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 29 October 2013 - 02:22 AM

You could extract the fat with a chloroform wash.

mmmm it sounds interesting... do you know how I might get the protocol?

Wash with cloroform, after of before add the laemli? / wash the whole protein extract?

Thanks a lot, this forum is really useful !!



#10 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,616 posts
118
Excellent

Posted 29 October 2013 - 03:30 AM

wash the whole extract, before adding laemmli buffer.

 

make sure you remove all of the organic phase or, at least, that you don't carry any over into the gel sample.


Edited by mdfenko, 29 October 2013 - 03:55 AM.

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

#11 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 30 October 2013 - 03:09 AM

wash the whole extract, before adding laemmli buffer.

 

make sure you remove all of the organic phase or, at least, that you don't carry any over into the gel sample.

Sorry for my question, but when you use a organic wash, I was thinking that my protein will go to the organic (chloroform) phase, isn´t  it?



#12 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,616 posts
118
Excellent

Posted 30 October 2013 - 04:06 AM

no. most proteins will remain in the aqueous phase.


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

#13 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 30 October 2013 - 05:13 AM

no. most proteins will remain in the aqueous phase.

Thanks a lot

I will try it just now!!!

Thanks!!



#14 Vemovied

Vemovied

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 11 November 2013 - 08:00 AM

You could extract the fat with a chloroform wash.

It was fantastic, the fat was removed completely, but I´m in trouble now for re suspend the protein, any suggestion for a good buffer?

Thanks so much!



#15 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,268 posts
213
Excellent

Posted 11 November 2013 - 08:18 AM

If you are simply doing an SDS gel, then you could dissolve in loading buffer (SDS or LDS). If that doesn't work, then dissolving in 4 to 8 M urea would surely work.






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

©1999-2013 Protocol Online, All rights reserved.