I want to extract and identify different types of protein in SDS that attached to the polyhydroxybutyrate granule which are intracellular protein, please suggest me any protocol that will be best for this experiment. Then how to purify those enzymes.
If you have access to mass spectrometry, isolate the granules, digest with trypsin, and determine the identity of the proteins using proteomics to compare the masses of the trypsin peptides to the proteome. If you have enough material, you may even be able to use MS to get some peptide sequences. If you decide to take this approach, avoid using detergents. They can be unfriendly to MS.
Once you identify the proteins, overexpress them in E. coli for purification.
If you have access to mass spectrometry, isolate the granules, digest with trypsin, and determine the identity of the proteins using proteomics to compare the masses of the trypsin peptides to the proteome. If you have enough material, you may even be able to use MS to get some peptide sequences. If you decide to take this approach, avoid using detergents. They can be unfriendly to MS.
Once you identify the proteins, overexpress them in E. coli for purification.
If you want to identify or quantify the proteins from granules, sonicate the granules in lysis buffer. Centrifuge to remove cell debris and concentrate the supernatant. It contains your proteins of interest (intracellular). Run on SDS-PAGE and visualize bands by staining with commasine blue. Follow in gel digestion protocol and analyse tryptic digested peptides by mass spectrometry. See my proteomics publications for details.
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