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21/09/2011 21:54:28
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topic:
mass compatible silver staining
bsengez
Posts 1
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Hi,
I used coomassie even colloidal coomassie staining for my 2D gels, but unfortunately I have poor results. Therefore, I prefer to use silver staining. Although I carried out a mass compatible silver staining protocol, I cannot get a good MS/MS result. I'm new in mass spec analysis and my problem is there are several mass compatible silver staining protocols and I cannot decide which to prefer. For instance, some protocols use formalin in each step but the others use it only in developing step. Some uses only EDTA in stopping step, the others use acetic acid. Therefore, I need people who have good mass spec analysis using silver stained spots.
Thanks in advance..
Burcu
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13/09/2010 13:49:46
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topic:
need help
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
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13/09/2010 08:38:18
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topic:
need help
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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07/09/2010 13:59:56
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topic:
need help
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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07/09/2010 13:23:54
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topic:
need help
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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26/08/2010 11:18:10
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topic:
need help
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.
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07/10/2009 15:37:31
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topic:
Editorial - flat-pack assembly and mass spec
FixingProteomics
Posts 2
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Have you ever wondered at the various types of screw heads you find in a typical piece of flat-packed furniture? When we're assembling our furniture, we typically don't care about the idiosyncrasies of screw types - after all, we're too busy figuring out the assembly manual. But when we are planning proteomics experiments, we do need to realize the properties of the technologies we're using. Continue reading...
edited by FixingProteomics on 07/10/2009
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30/04/2009 14:31:44
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topic:
Editorial: Sample preparation - key to proteomics?
FixingProteomics
Posts 2
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"Sample preparation - a necessary evil or the key to proteomics?
Over the years, the main focus of proteomics has been on technological aspects such as capacity and dynamic range of 2D gels, masspectrometry resolution and sensitivity. Enormous amounts of money and effort have been spent on this work while the softer side of proteomics; study design, sample preparation and reproducibility have been left out in the cold. The old saying "garbage in, garbage out" holds as true for proteomics as other parts of life, no matter how many millions of Euros you spend on for instance mass spectrometers. In order to fix proteomics and release its full inherent potential these softer aspects must be allowed a place on centre stage."
Continue reading...
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