Western blot sample preparation occurs before SDS-PAGE; it is the start of Western Blot. The sample exctract can be prepared from cell culture or tissues by mechanical crushing like homogenization and sonication methods or high pressure disruption, and then they are further lysed with some cell lysis buffer to make sure the maximum percent of proteins have been extracted. Extraction is always performed on ice or at 4°C at all times to prevent protein degradation.
Choosing the right lysis buffers for your samples is the first step of your Western Blot to success. If you use improper lysis buffers, your protein of interest may not be fully extracted or cannot be barely extracted at all. So, based on the expression location of your protein of interest, you can select the proper lysis buffers and avoid this problem.
|Protein location||Buffer recommended|
|Nuclear / Mitochondria||RIPA, or nuclear / mitochondria fractionation for increased protein of interest concentration|
|Cytoplasmic (soluble)||Tris-HCl buffer|
|Cytoplasmic (cytoskeletal bound)||Tris-Triton buffer|
|Membrane bound||NP-40, RIPA or Tris-Triton buffer|
Protease and phosphatase inhibitors are added to protect the proteins from digestion by protease that leaked out during sample preparation.
|Inhibitor||Structural formula||MW (Da)||Specificity of Inhibitor||Solubility (Solvent)||Working concentration*|
|Aprotinin||-||6512||Chymotrypsin, plasmin, trypsin, and kallikrein||10 mg/mL (H2O)||0.01 – 0.3 µM|
|Chymostatin||604.7||Serine proteases||20 mg/mL (DMSO)
20 mg/mL (glacial acetic acid)
|10 – 100 µM|
|Bestatin||308.4||Aminopeptidase B, Leucine aminopeptidase and Aminopeptidases N||5 mg/mL (methanol)
20 mg/mL (1 M HCI)
1 mg/mL (0.15M NaCl)
|E-64||357.4||Cysteine proteases||25 mg/mL (DMSO)
20 mg/mL (aqueous buffers)
|1.4 – 28.0 mM|
|Leupeptin||426.6||Serine, plasmin, porcine
kallikrein and cysteine proteases
|1 mg/mL (H2O)||1 µM|
|PMSF||174.2||Cysteine and serine proteases||18 mg/mL (methanol)||0.1 – 1.0 mM|
|Pepstatin A||685.9||Aspartic proteases||1 mg/mL (ethanol)
1 mg/mL (methanol)
300 μg/ml (6 N acetic acid)
|1 – 20 µM|
|EDTA||372.24||Metalloproteases||0.5 M (H2O)||0.5 – 1.3 mM|
|AEBSF•HCl||239.7||Chymotrypsin, kallikrein, plasmin, thrombin and trypsin||200 mg/mL (H2O)||0.1 – 1 mM|
In order to ensure equal loading of each lane, determination of protein concentration is inevitable. Biochemical analysis of proteins relies on accurate quantitation of protein concentration. There are some commonly used protein assay methods to determine protein concentration in biochemical laboratories, including the Lowry assay, the Bradford assay, the BCA assay, and UV spectroscopy. According to your experiment conditions, you can perform one assay to determine protein concentration.
|Sample composition||Protein assay not recommended||Protein assay recommended|
|Arginine-rich protein||Bradford assay||Lowry, BCA or UV assay|
|Cysteine-rich protein||BCA assay||Lowry, UV or Bradford assay|
|Tryptophan- or tyrosine-rich
(no extinction coefficient)
|UV assay||Lowry, BCA or Bradford assay|
After determination of protein concentration, samples are diluted in gel loading buffer, also called 2x Laemmli sample buffer. This buffer contains glycerol helping the samples sink easily into the wells of the gel, and a tracking dye (bromophenol blue) which reaches the bottom of the gel indicating the end of electrophoresis.
Before loading into PAGE gels, samples are heated for either 5 minutes at 100°C, or 10 minutes at 70°C to aid in the denaturation. By now, samples can be loaded into gels immediately, or placed at 4°C or -20°C for later analysis.
|1||Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout).|
|2||Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.|
|3||Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.|
|4||Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail).|
|5||Incubate for 30 minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.|
|6||Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C.|
|7||Measure the concentration of protein using a spectrophotometer.|
|8||Using the formular "Concentration=mass / volume", determine the volume of protein extract to ensure 50 μg in each well.|
|9||Add 5 μL sample buffer to the sample, and make the volume in each lane equalized using double distilled H2O (dd H2O). Mix well. (Tip: Total volume of 15 μL per lane is suggested).|
|10||Heat the samples with dry plate for 5 minutes at 100°C.|