WB Technical Resources

Protocols, optimization tips,
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Troubleshooting guides

Troubleshooting guides

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To extract protein for the western blot analysis, the first step is to prepare for the lysate from samples. Cell culture and tissue are the most common samples to start with.

  1. Protein Extraction

    1. Extraction from Cell Cultures
      • Culture cells in the cell culture dish until 80% confluency.
      • Place the dish on ice and rinse cells in ice-cold PBS buffer (10 mM Na2HPO4 and 1.8 mM Na2H2PO4 with 0.2% Tween 20; pH 7.4).
      • Aspirate the PBS and add ice-cold lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl and protease inhibitors).
      • Scrape adherent cells off the dish using cold plastic cell scrapper or digest cells with 0.05% trypsin cells with PBS wash. Be as gentle with cells as possible to avoid inducing cell stress pathways.
      • Centrifuge the cells at 3,000 rpm at 4°C for 2-3 min.
      • Remove the supernatant and wash it 2X with ice-cold PBS buffer.
      • Gently transfer the cell precipitate into an ice-cold tube.
      • Add Mammalian Cell Protein Extraction Reagent (Boster Catalog # AR0103) into the tube (v/v: 6/1: extraction reagent/cell precipitate) and resuspend vigorously.
      • If the above solution is murky, sonicate it for 10-15 sec to break up the proteins.
      • Lyse the cells in RIPA lysate buffer on ice for 4-5 hours.
      • Sonicate and lyse again if the cell solution remains murky.
      • Centrifuge at ~10,000 rpm and 4°C for 10 min. The centrifugation force and time can be varied depending on the cell type.
      • Discard lipid (at top) and cell debris (at bottom) by aliquotting the solution in the middle to a fresh tube and keeping the tube at -20°C.
    2. Extraction from Tissues
      • Place surgically resected tissues in pre-cooled (4°C) normal saline. Make sure to wash off any blood from the tissues.
      • Cut the tissue into small pieces (0.1 g to 1 g each).
      • Add Broad Spectrum Protease Inhibitor Cocktail (Boster Catalog # AR1182).
      • Add 10 mL Mammal Tissue Protein Extraction Reagent (Boster Catalog # AR0101) per 1 g of tissue.
      • Mince the tissue and place the minced tissue in tissue homogenizer.
      • Add ice-cold lysis buffer (For a 5 mg piece of tissue, add 300 µL of buffer. Buffer volume should be determined in relation to the amount of tissue present).
      • Lyse the tissue homogenate on ice for 4-5 hours or at 4°C (high speed) for 5 min.
      • If necessary, sonicate until no tissue chunks remain.
      • Centrifuge at ~10,000 rpm and 4°C for 10 min. The centrifugation force and time can be varied depending on the sample type.
      • Discard lipid (at top) and cell debris (at bottom) by aliquotting the solution in the middle to a fresh tube and keeping the tube at -20°C.
  2. Protein Quantitation Assay

    The test tube protocol for our BCA (Bicinchoninic Acid) Protein Assay Kit (Boster Catalog# AR0146) is described here. Refer to our datasheet for the microplate protocol as appropriate.

    1. Reagent Preparation
      • Reconstitute the albumin standard ampules (BSA) with 0.9% NaCl or PBS to create a working solution of 2000 µg/mL (Tube A).
      • Mix thoroughly 50 mL BCA Reagent A with 1 mL BCA Reagent B (i.e., Diluent).
      • Prepare the diluted BSA standards by mixing the diluent and BSA as follows:
      Tube Diluent Volume (µL) BSA Volume (µL) BSA Concentration (µg/mL)
      A 0 600 (From Tube A) 2000
      B 100 300 (From Tube A) 1500
      C 300 300 (From Tube A) 1000
      D 200 200 (From Tube B) 750
      E 300 300 (From Tube C) 500
      F 300 300 (From Tube E) 250
      G 300 300 (From Tube F) 125
      H 400 100 (From Tube G) 25
      I (Blank) 300 0 0
    2. Quantitation Assay (For the test-tube protocol, ratio of sample to working range is 1:20).
      • Pipette 0.1 mL of each standard and unknown sample replicate into an appropriately labeled test tube.
      • Add 2.0 mL of the working range (WR) to each tube and mix well.
      • Cover and incubate tubes with one of the protocols:
        • Standard Protocol: 37°C for 30 min (WR: 25 to 2,000 μg/mL).
        • RT Protocol: Room temperature for 2 hrs (WR: 25 to 2,000 μg/mL).
        • Enhanced Protocol: 60°C for 30 min (WR: 5 to 250 μg/mL).

      Note: Increasing the incubation time and temperature can increase the net 562 nm absorbance for each test and decrease both the minimum detection level of the reagent and the working range of the protocol. Use a water bath to heat the tubes for either Standard (37°C incubation) or Enhanced (60°C incubation) Protocol. Using a forced-air incubator can introduce significant error in color development because of uneven heat transfer.

      • Cool all tubes to room temperature
      • With the spectrophotometer set to 562 nm, “zero” the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples within 10 min.

      Note: Because the BCA Assay does not reach a true end point, color development will continue even after cooling to room temperature. However, because the rate of color development is low at room temperature, no significant error will be introduced if the 562 nm absorbance measurements of all tubes are made within 10 min of each other.

      • Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm absorbance measurement of all other individual standard and unknown sample replicates.
      • Prepare a standard curve by plotting the average blank-corrected 562 nm measurement for each BSA standard vs. its concentration in μg/mL. Use the standard curve to determine the protein concentration of each unknown sample.