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. 293 cell research: The target protein is expressed in the cytoplasm and nucleus.The protein expression can be validated by extracting the total protein of the tissue sample. Mouse liver tissue research: The expression level of target protein is very high.The research on target protein includes the expression level, location and unique properties. Sample type, amount and the purpose of western blot should be confirmed first. Keywords: Western Blot Sample Preparation, Protein Extraction for Western Blot, Western Blotting Techniques, Western Blot Analysis 1. Protein extraction is the simplest extraction technology. For HRP conjugated antibodies, use an enhanced chemiluminescence (ECL) kit and follow manufacturer’s guidelines.Abstract: Western blot sample preparation plays an important role in obtaining a satisfied western blot band. For fluorescently labelled antibodies, use a fluorescence scanner, as per the manufacturer’s instructions. Transfer membrane onto cling film, protein side up.ĭetection will depend on the conjugation of your primary or secondary antibody. Incubate the membrane with secondary antibody at appropriate concentration in blocking buffer and incubate for ~1 hr on shaker at room temperature.Incubate the membrane with primary antibody at appropriate concentration in blocking buffer for ~1-2 hrs on shaker at room temperature.Wash the membrane with wash buffer for 5 mins on a shaker at room temperature.PVDF membrane must be reactivated by soaking in methanol for 1 min.) Incubate the membrane in blocking buffer at 4☌ overnight.Remove Ponceau S stain using 0.1 M sodium hydroxide solution.Note: Use a pencil to label tracks on the membrane and indicate which side the protein is on. Cut the membrane for staining with different antibodies.Remove excess stain by rinsing membrane in 5% acetic acid solution.Incubate membrane using Ponceau S solution for 10 mins to visualise and confirm protein transfer.Note: Transfer times depend on protein size. Assemble the gel, membrane, filter paper and pads in the blot transfer cell (pad/paper/gel/membrane/paper/pad).Note: Nitrocellulose membrane does not need activating. Activate PVDF membrane in methanol for 1 min before equilibrating in blotting buffer for 5 mins.Incubate gels in blotting buffer for 10 mins.Blocking Buffer: 5% BSA, 0.05% Polysorbate 20 in PBS.Wash Buffer: 0.05% Polysorbate 20 in PBS.
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