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Protein Sample Preparation

Initial protein extraction is often based on buffers containing mild non-ionic detergents, various salts, stabilizing agents, chelating agents, reducing agents, and protease inhibitors.

The choice of protease inhibitor may be critical to maintaining yield, structure, and function. Nucleases can be used to remove nucleic acids, and care should be taken to remove contaminants, including detergents that might interfere with downstream applications.

The preparation of protein samples is critical to the success of analytical methods such as mass spectrometry, 2-D gel electrophoresis, 1-D SDS-PAGE, trypsin digestion, and chromatography.

 

Protein Enrichment & Sample Concentration Cell Lysis & Protein Extraction Sample Clean-Up Protein Quantitation

Capture and enrichment of a specific target protein helps to enhance detection signal strength. This strategy is effective both for proteins from natural sources as well as recombinant proteins.

GE Healthcare offers an extensive range of affinity-based products for protein enrichment. For convenient capture of tagged recombinant proteins, use our affinity chromatography media, available in both column and plate format. We also offer Mag Sepharose magnetic beads for tagged protein purification and enrichment of phosphopeptides.

Concentrating the sample is one effective way to load more protein per analysis. With Vivaspin™ Sample Concentrators, up to 30-fold concentrations possible and recovery of the target protein typically exceeds 95%.

In general, gentle methods are employed when the sample consists of easily lysed, cultured cells or blood cells, whereas more vigorous methods are employed for the disruption of more robust bacterial or plant cells, or mammalian cells embedded in connective tissue. Protease inhibitors should be added to lysis buffers to prevent degradation of proteins due to the release of endogenous proteases as cells are broken down.

We offer a range of kits and buffers for lysis and protein extraction to suit different needs, depending on cell type and the overall purpose of your experiment. Refer to the Cell Lysis and Protein Extraction table or browse all of our Lysis and Protein Extraction products.

Compounds such as nucleic acids, polysaccharides, and salts may interfere with downstream applications such as electrophoresis. Sample cleanup may thus improve the quality of your downstream results. Desalting based on gel filtration may be achieved in a single step, with the added advantage that the sample is eluted into an appropriate buffer, so that your sample is ready for the next step in your workflow.

DNase may be used to counter problems with viscosity caused by the release of nucleic acids. Plasma or serum samples contain proteins such as albumin and IgG that can obscure the signals of less abundant proteins. HiTrap Albumin & IgG Depletion prepacked columns are designed to deplete samples of these potentially problematic proteins, removing more than 95% and 90% of albumin and IgG, respectively.

Quantitative analysis requires that all lanes in a gel are loaded with the same amount of total protein. Several spectrophotometric methods are routinely used to determine the concentration of protein in a solution but none are particularly convenient. 2-D Quant Kit, despite its name, is well-suited to the accurate determination of protein concentration for all electrophoresis applications.