Western Blot Protein Sample Preparation Protocol

Information on Western Blot Sample Preparation. Cell Lysis and Quantitation Protocol for Western Blotting.

Protein Sample Preparation Protocol for Western Blot

Cell Lysis and Quantitation Protocol for Western Blotting

• Keep protein samples on wet ice during the whole protein sample preparation isolation and western blot procedure.
• Lyse cells in buffer or detergent
• Selection of cell lysis buffer depends on protein solubility,
• Cell lysis buffer may contain proteases inhibitors, phosphatase inhibitors, and other inhibitors.
• Sonicate sample to break up genomic DNA.
• Centrifuge sample at 10,000 rpm for 5 minutes.
• Transfer supernatant to a new tube.
• Quantitate protein concentration by Pierce BCA protein assay kit. Determine the protein concentration (Bradford assay, A280, or BCA).
• Set up the following mix (20ul total volume)
• Sample Volume for 5-20ug protein__ H20 to 10ul 2X SDS sample buffer x y 10ul
• Take x µl (= y µg protein) and mix with x µl of 2x sample buffer.
• Boil samples in heat block for 2 minutes - 5 min.

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