Soft tissues can be processed by a tissue grinder which consists of stainless steel or glass hammer and a glass tube. Tissue grinding usually requires for homogenization. Before extraction, using PBS to wash the blood to prevent the influence of IgG in the blood on the following experiment. ![]() If the taken tissues are not used at once, they should be cryopreserved in liquid nitrogen or refrigerator at -80℃. Finally, take organs like heart, spleen, kidney, brain etc. Then, take reproductive related tissues (e.g. stomach, large and small intestine, liver, pancreas etc) and macrophage rich tissue first. Take the digestive system related tissues(e.g. The trypsin digestion technique is not suggested to use for collecting cells in membrane expressed proteins. Proteins in adherent cell can be directly extracted in the cultivating container. Suspension cell can be centrifuged at 500Xg and then collected for protein extraction. 1-2ml extraction reagent is sufficient for per 100 mg tissue.īefore extraction, using PBS to wash cell samples twice to reduce the interference of culture medium on samples. To efficiently extract protein, 100-200μl extraction reagent is usually instilled into each well of 6-well plate. Thus, the protein should be extracted in the specific region, following your experimental purpose. The protein expression level probably varies in different regions. The brain is also divided into different regions, belonging to non-homogeneous organs. ![]() renal tissue consists of cortex and medullar. Phosphatase inhibitor should be added for validating phosphorylated protein.ĭuring extracting tissue proteins, the position of organ tissues should be noticed. Two kinds of inhibitors prevent the target protein from degradation. Protease and phosphatase inhibitors assist to extract proteins in tissues, cells or subcellular fractions. However, their basic components are nearly cell lysates. There are various protein extraction reagents in the market. Furthermore, The protein should be maximally extracted by the proper ratio. The protein extraction is based on the full protein profile. Then, cytoplasmic and nucleolus proteins are detected respectively. Target protein expression level in different components can be compared by separating the cytoplasm and nucleus of the cell sample.
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