Technique / Proteomics and protein biochemistry


Proteomics protocols and troubleshooting



Proteomics protocols and troubleshooting

Protocols for some proteomics application (protein extraction, IEF, SDS-PAGE, 1D and 2D PAGE, in gel staining and protein detection). It also includes pictures of different types of electrophoresis, proteomics troubleshooting and list of vendors for proteomics study. (Biocenter Oulu, University of Oulu, Finland)

Content:

1. Sample preparation
1.1. Escherichia coli
1.2. Saccharomyces cerevisiae
1.2.1. Whole intracellular extract
1.2.2. Mitochondria
1.3. Mouse
1.3.1. Kidney
1.3.2. Bones (calvaria and femur)
1.3.3. T-cells from spleen
1.3.4. Fibroblast cell line NIH3T3
1.4. Human
1.4.1. Serum
1.4.2. Tooth pulp tissue
2. IEF (Isoelectric focusing)
2.1. In-gel rehydration
2.2. Sample cup loading
3. SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
3.1. Casting the SDS gels
3.1.1. 2-D SDS-PAGE
3.1.2. 1-D SDS-PAGE
3.2. Electrophoresis
3.2.1. 2-D SDS-PAGE
3.2.2. 1-D SDS-PAGE
4. Protein detection
4.1. Silver staining
4.2. Western Blotting
5. Protein identification (mass spectrometry)
6. Troubleshooting
7. Links to other protocols
8. References
9. Chemicals

Last update 24-Mar-2005, Rating of 6 votes.

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By zhangwei on 02-Sep-2007
high molecular weight proteins ?>100kDa?
2D is suitable for separating 10 to 100kDa of proteins
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By Sailesh on 29-Aug-2007
I m doing my PhD in ICGEB, in proteomics, my work involves 2d PAGE and MS.
my problem is that, when I perform 2 D PAGE, the high molecular weight proteins are not visible at all.and the upper portion of the gel is completely clear.this is not the matter of over run as the dye front is visible at the end of the gel.and the lower portion of the gel is perfect and the spots are distinctly visible.
can some one please suggest me what might go wrong as soon as possible.
thanky you
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By Binesh on 18-Sep-2006
I am a post graduate from India and I would like to be my PhD and career in Proteomics. If anybody is there to help me in this topic please send me the relevent e-mail id so that I can be frequently in contact with you with my doubts and such things. I will be thankful to you for ever if you do this favour for me. Thanking you..........
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By ademola on 17-Jul-2005
good,iam impressed.
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By deepak gupta on 15-Jun-2005
very nice and useful information
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By sreejith on 24-Mar-2005
proteomics
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