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2-D Gel Electrophoresis Forum 2-D Gel Electrophoresis Forum. A forum dedicated to two dimensional gel electrophoresis, discussion about systems, and troubleshooting its problems.


2D troubleshoot

2D troubleshoot - 2-D Gel Electrophoresis Forum

2D troubleshoot - 2-D Gel Electrophoresis Forum. A forum dedicated to two dimensional gel electrophoresis, discussion about systems, and troubleshooting its problems.


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  #1  
Old 08-22-2008, 07:53 AM
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Default 2D troubleshoot



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

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File Type: jpg 2-d-troubleshoot.jpg (25.7 KB, 116 views)

Last edited by admin; 08-26-2008 at 10:05 PM.
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Old 08-23-2008, 12:28 AM
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Default Re: 2D troubleshoot

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?
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Old 08-23-2008, 12:29 AM
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Default Re: 2D troubleshoot

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

cheers
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Old 08-23-2008, 05:35 AM
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Default Re: 2D troubleshoot

unluckily i lost the 2D pic og extract but there is my pic of plasma 2D showing same kind of results
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Old 08-24-2008, 10:43 AM
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Default Re: 2D troubleshoot

Hello Aaina, yes that can help you can Go Advanced and Upload Attachment with browse.
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Old 08-25-2008, 02:46 AM
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Default Re: 2D troubleshoot

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.
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Old 08-25-2008, 08:30 AM
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Default Re: 2D troubleshoot

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
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Old 08-26-2008, 02:29 AM
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Default Re: 2D troubleshoot

yes i did it. thanx
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Old 08-26-2008, 05:51 AM
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Default Re: 2D troubleshoot

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
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Old 08-27-2008, 02:31 AM
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Default Re: 2D troubleshoot

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.

Last edited by Aaina; 08-27-2008 at 02:37 AM.
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