Proteomics/Protein Sample Preparation/Sample Preparation for Chromatography
Sample Preparation for Chromatography
[edit | edit source]
Solubilization and Solution Preparation
[edit | edit source]For High Performance Liquid Chromatography (HPLC), it is important that the sample solution (mobile phase) be free of particulate matter. Due to the sensitive nature of a HPLC column and the extreme pressures involved, such contaminants can be extremely damaging and contaminant elimination is very important. Each specific liquid chromatography technique requires specialized solutions. Reversed-phase chromatography requires polar organic solvents, while normal phase chromatography can use inorganic solvents such as water. One of the most common organic solvents is acetonitrile. This polarity gives the solvent the ability to elute proteins out of a reversed phase column, and it is also easily coupled to mass spectrometry. Also, solution buffers must contain high quality pH stabilizers such as acetic acid (acid) and sodium acetate (base). Salt buffers such as Potassium Chloride are used to produce elution conditions in ion-exchange chromatography, and also must be of very high purity. A list is given below of common chromatography reagents:
- Buffer reagents
- Acetic Acid
- Phosphoric Acid
- Triethylamine
- Sodium Acetate
- Ammonium Acetate
- Potassium Phosphate
- Sodium Bicarbonate
- Sodium Chloride
RIPA is radio immunoprecipitation Assay the buffer can be used to solubilize proteins in a cell. Analysis shows that RIPA dissolves some membrane-bound proteins and most intracellular proteins. RIPA is not to be used to find protein-protein interactions because this buffer breaks the bonds of the protein-protein formations.
Recombinant Techniques for Affinity Chromatography
[edit | edit source]Affinity chromatography is a protein separation technique where substrates with affinities for specific proteins are used in the solid phase of a chromatography column. One highly specific technique is to use recombinant technology to isolate proteins expressed from recombinant DNA. If a gene is inserted into a cloning vector for the purpose of fermenting large amounts of a protein, the gene can be altered to include additional amino acids in the protein. This subsequence can then be utilized as a binding site for the stationary phase substrate. Two examples of this are the additions of histidine residues that bind covalently to certain metal ions and the addition of glutathione S-transferase[1] protein that will bind with glutathione.
References
[edit | edit source]http://en.wikipedia.org/wiki/Affinity_chromatography
Single-step purification of bacterially expressed polypeptides containing an oligo-histidine domain. Gene. 1992 Feb 1;111(1):99-104.
Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem. 1993 Apr;210(1):179-87.
Articles Summarized
[edit | edit source]Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers
[edit | edit source]Ngoka, Lambert CM; Proteome Science 6: 30. pg 1-21; Published online 2008
The focus of this paper is to prepare a protein sample from cancer cells using the widely established RIPA method. RIPA buffer does this by taking proteins from there native insoluble state and solubilizing them into a separable sample. It also separates the proteins from salts and other contaminants making it a reliable sample prep tool. A problem with using just one sample buffer is that the buffer may not have solubilized all the proteins from the sample cell. This experiment, in addition to RIPA, also uses urea to extract proteins missed by RIPA. Tumor cells with varying origin characteristics were subjected to RIPA analysis and urea analysis. It was concluded that using the different methods each cell yielded some of the same proteins and many unique proteins. With the RIPA analysis many intracellular proteins and proteins with small molecular weights were readily soluble. Some Other Proteins would bind weakly to the buffer following for solubility. The results for the urea analysis proved somewhat opposite, this test would solubilize extracellular matrix proteins and proteins with high molecular weights. This is not to say that neither protein yielded the same results, from the paper it can be witnessed there is a big overlap of the same proteins found with different analysis. When making a protein sample or finding novel proteins experimenters should use multiple isolation techniques to obtain the greatest population of proteins available.
New Terms
plasmogen activators- molecule used to disassociate blood clots.
angiogenesis - blood vessel genesis and progression. (dictionary.com)
In the extraction process of proteomics it is useful to use more than one extraction buffer to obtain different libraries of proteins.
Websites Summarized
[edit | edit source]Immunoprecipitation
[edit | edit source]Summary
Immunoprecipitation has several steps, the first is to lysis the cell and obtain a protein mixture. It is then a good idea to clean the cell of all non-specific proteins. Following this cleaning you want to add the anti-bodies to precipitate the targets. Then you can isolate your sample for further analysis. Some lysis buffers are RIPA and NP-40, RIPA disrupts protein-protein interactions and NP-40 is used to identify protein-protein interactions. Success of this technique depends on the affinty binding of the protein-antibody formation.
Immunoprecipitation is a method of protein extraction, usually radio-labeled this technique can obtain the molecular weights and quantity of protein samples. Samples are extracted using anti-body- protein complexes. Immunoprecipitation can also be used to determine protein- protein interactions, rate of synthesis of a protein, and can concentrate small levels of proteins.
New Terms
pre-clearing - A step in imuunoprecipitation that lowers non-specific proteins.
mono-clonal antibodies - antibodies cloned to produce high volume populations of themselves. (dictionary.com)
Immunoprecipitation is widely used in sample preparation of proteins, it yields samples with acceptable concentrations and accurately targets specific proteins.