2D troubleshoot

[Aaina]

Hi.

I m working on human protein extract from tissue samples but after running second dimesion no separation has occured. I ran the same sample in duplicate but no resolution. there are a few cm lines on the gel everywhere each time. plz help me

Edit by admin: Here is an image of the 2D gel

[dave2006]

hi there,
the second dimensions on a 2D gel separates your proteins by charge. so is there anything that may have affected this in your method?

[admin]

hi Aaina,
welcome to the forums. Can you post a image of your gel, that would help to solve this.

cheers

[Aaina]

unluckily i lost the 2D pic og extract but there is my pic of plasma 2D showing same kind of results

[admin]

Hello Aaina, yes that can help you can Go Advanced and Upload Attachment with browse.

[Aaina]

sorry yar

My image is in Bit map format and is not converting in any other format, so ca not be loeaded its word file is too heavy to be loaded. i m so helpless.

[admin]

Hello,
I would be willing to take a look and even post it here for others to help you if you simply email your bitmap to webmaster[at]molecularstation.com

cheers

[Aaina]

yes i did it. thanx

[deepee]

Hi Aaina, could you give us a quick run down of your protocol too please? What type of tissue are you extracting protein from and how? How did you separate your sample and how much protein did you run. The more information you can give the better, especially if you can't attach a picture.
Also, after you have got help from this forum, will you have enough sample available to repeat the experiment? 2D gels are notoriously difficult so don't worry too much that your first attempt hasn't worked too well.
Thanks, and good luck.
deepee

[Aaina]

i simply ran human plasma by adding sample in rehydrartion buffer, incubationg IPG strip in it overnight and ran it for 20,000kvh. before second dimension, equilibrated the strip in DTT and iodoacetamide buffer successively, Did second dimension with the usual SDS protocol. For tissue sample, i did urea lysis and got similar kind of results.