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26/08/2010 11:18:10
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pushpa
Posts 2
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Hi..Here is the attached gel image. Can u help me find out wat is the cause of the smear on the gel. The condition of the gel is:
1) Sample: Depleted Plasma ( The plasma has been depleted by using Hi Trap Albumin & IgG removal Kit from GE)
2) pH of the IPG strip is pH 3-10 NL
3) Its a precast gradient gel ( 4-15%) (Bio-Rad)
4) Protein concentration:100ug
5) staining method: Coomassie brilliant blue
4) First dimension steps:
The steps were:
Step 1: 150V, Linear, 15minutes.
Step 2: 500V, linear, 30 minutes
Step 3: 100V, linear, 30 minutes
Step 4: 4000V, linear, 1hr 30 minutes
Step 5: 4000V, Rapid, Vhrs:10000
Step 6: Holding steps.
Hope to get the reply asap.
Thanx alot.
Regards,
Pushpa.
Attachments:
gel image.JPG
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07/09/2010 13:23:54
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hovinsj1
Posts 1
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Your gel is typical for a serum sample. Serum is probably the most difficult sample to run on a 2D gel. How did you prepare the sample after depletion? The standard lysis buffer for 2D gels is 7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1-2% Pharmalytes. I would suggest another gradient for the 2nd dimension as well, we were very successful with a 10-15% gradient (large gels). What is actually the length of your 1st dimension, is it a 7 cm strip. If so, you can also reduce the total amount of kVh.
sjouke
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07/09/2010 13:59:56
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gucio1
Posts 1
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my guess is that the smear comes from overload of Albumin... maybe the depletion did not work very well.
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08/09/2010 11:00:12
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Guest
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Hi...It is 7cm strip...Can you kindly please explain to me about the reducing the kVh...seems not clear with your sentence..Another thing about the gel is I couldnt find any companies providing the gradient gel other than Bio-Rad...this is because in my lab I dont have the gradient gel maker...so, I got no options to get the larger gels with different gradient....Do you have any idea about it? I do use the typical lysis buffer as you mentioned. I plan to reduce the amount of protein. I got to tell you that after depletion of albumin & IgG, i do desalting the samples and after that I send the samples for freeze drying. Then I perform with protein quantification and followed by rehydration of the strip , first dimension, equlibirium step and then second dimension and staining. Thanks alot for your reply.
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13/09/2010 08:38:18
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Reiner Westermeier
Posts 1
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Why do want to separate the 2nd dimension in such a gradient? You can get very good results in homogeneous gels. A 7 cm gel with pH gradient 3-10 cannot resolve very much, it is sensitive to overloading. Obviously there is s till a lot of albumin in the samples...
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13/09/2010 13:49:46
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pushpa
Posts 2
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I did try with homogeneous gel and i dont see any good separation of the protein. Thats why one of the Field Specialist Applicant advise me to use the gradient gel.What sort of pH gradient you would recommend if you saying pH 3-10 cannot resolve very much? I did attached the gradient gel with pH4 -7 which was checked for the depletion kit? From this image I dont see any contamination with Albumin and IgG. Thats why I hold on the same method.
Thank You.
Regards,
Pushpa
Attachments:
spots.jpg
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