Runing problems - (Oct/24/2013 )

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

Hey Vemovied,

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

were you using adipose tissue in the past?

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

Vemovied on Thu Oct 24 12:51:55 2013 said:

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

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.

mdfenko on Mon Oct 28 13:24:56 2013 said:

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?

You could extract the fat with a chloroform wash.

phage434 on Mon Oct 28 23:54:59 2013 said:

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 !!

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.