Pure deionized or double distilled water should be used when required for all solutions. Always remove the stacking gel and trim the gel at the bottom by at least 1 mm. Gels are incubated on a rocking platform or an orbital shaker or at a moderate speed of 40–70 rpm and should float freely in solution. Dishes should be cleaned with 70–100% ethanol or methanol followed by a water rinse. Gel staining is generally accomplished in covered polycarbonate or polypropylene dishes. 1.1 Universal PracticeĬommon protocols of all gel-staining methods require good laboratory practices. Posttranslational modifications can be measured by specialized staining formulations and protocols that target the modified functional groups from a background of total protein. Protein detection in SDS-PAGE can be done by noncovalent postelectrophoresis staining with organic dyes or by metal deposition techniques or by labeling samples by covalent modification with a fluorescent dye prior to running gels. A detection method that accurately assesses the total protein profile is generally sufficient for a general purification protocol. The common factor in both these methods is the use of colorimetric and fluorescent stains for total protein detection for further downstream applications ( 6– 9). One-dimensional SDS-PAGE protein analysis is the most common method to separate and detect proteins but 2D gel electrophoresis format is preferred for analysis of complex protein mixtures. A typical visual example of showing protein purification from a mix involves showing fewer number of proteins with each step along with enrichment of the protein of interest ( 2– 5). Here, we focus on protein detection after separation by electrophoresis, highlighting on most popular methods for detection of both total protein and most frequently encountered protein posttranslational modifications such as phosphorylation and glycosylation ( 1). Protein posttranslational modifications such as phosphorylation and glycosylation can be reliably determined with several fluorescence-based protocols. Detection of separated proteins is carried out by colorimetric or fluorescent staining. This process separates proteins from a mixture based on size. The solution to check purity issues can be tackled by running a denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel. The most important hurdle that any scientist faces on a daily basis is obtaining proteins of high purity and decent yield.
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