Western Blotting Protocol
Provided by Biotium
Western blotting involves detection and visualization of specific proteins within a sample by multi-step laboratory process starting with gel-electrophoresis of a prepared protein sample to separate the protein mixture based on denatured peptide length and aggregate charge.
Size/charge-separated proteins are then transferred (blotted) to a thin membrane, also using electrophoresis, for specific detection by primary antibody affinity, visualization using a secondary antibody conjugated to fluorophore dye or chemiluminescent enzyme system.
Numerous variations of this protocol exist based on specific reagents different electrophoresis methods, primary and secondary antibody conjugates, and light detection equipment and methodologies.
Fluorophore Dye - Mix-n-Stain CF Dye Antibody Labeling Kits from Biotium
Chemiluminescent Enzyme System - Mix-n-Stain HRP, R-PE and APC Antibody Labeling Kits from Biotium.
Overview of Steps
- Sample Preparation: Cell lysis and denaturation of proteins.
- Gel Electrophoresis: Separation of proteins based on size and charge.
- Membrane Transfer: Transfer of size/charge-separated protein bands to a thin membrane.
- Membrane Blocking: Incubation of protein-transferred membrane with a casein, serum-albumin, fish gelatin, or commercially available solution to reduce noise from non-specific binding
- Primary Antibody Incubation: Binding of primary antibody to specific target antigen.
- Secondary Antibody Incubation: Binding of conjugated secondary antibody to primary antibody.
- Detection and Readout: Exposure of prepared membrane to light-sensitive film or CCD camera for visualization of specific proteins.
Reagents and Buffers
- Lysis Buffer (RIPA)
- 5X Loading Buffer (Laemmli)
- Running Buffer
- Gel Stain Solution
- TBST Wash Buffer
- PBS Wash Buffer
- Blocking Solution
- Primary Antibody
- Secondary Antibody Conjugate
- Collect appropriate amount of bacterial or tissue culture cells, spin samples on a tabletop centrifuge for 2 min at 15,000 RPM, and discard cell culture supernatant. Maintain all cell preparations on ice.
- Wash cells in cold PBS, discard supernatant.
- Add appropriate amount of cold Lysis Buffer. Sonicate each sample thoroughly to shear DNA and complete cell lysis.
- Incubate samples at 95 C for five minutes, then chill on ice.
- Re-centrifuge lysed samples and transfer supernatant to fresh tubes.
- Dilute to appropriate total protein concentration for loading.
- Add 5X Loading Buffer to each sample and load amount appropriate for size of polyacrylamide gel (typically 5-50 uL of 5-50 ug/mL protein concentration for “mini” sized gels). Save at least one lane to load a mix of molecular weight markers.
- Pre-run the gel at low voltage (30-50V) for 5-10 min to stack proteins.
- Increase voltage to 100-200V and run for 30-90 min until the Loading Buffer dye front is near the bottom of the gel.
- Remove gel from electrophoresis apparatus and transfer to a dish with Transfer Buffer for 10-20 min. Add an appropriate amount of Transfer Buffer to completely immerse the gel.
- Assemble the gel into a gel transfer sandwich apparatus (anode – blotter paper - gel – membrane – blotter paper – cathode).
- Load sandwich apparatus into transfer apparatus with Transfer Buffer, run overnight at 4C at constant current (10-20 mA).
- Remove blot from transfer apparatus and 3 X 5 min with TBST on an orbital shaker.
- Discard wash solution and add sufficient Blocking Solution to thoroughly cover membrane.
- Incubate on an orbital shaker for 1 hr at RT.
Primary Antibody Binding
- Discard Blocking Solution from membrane.
- Dilute appropriate amount of primary antibody into Blocking Solution.
- Add diluted primary antibody to membrane. Incubate overnight on an orbital shaker at 4C.
Secondary Antibody Binding
- Discard primary antibody solution. Wash membrane 5 X 5 min with TBST.
- Dilute appropriate amount of secondary antibody in Blocking Solution and add sufficient volume to cover surface of membrane.
- Incubate for 1-3 hrs at RT on an orbital shaker.
- Wash 5 x 5 min with TBST.
Detection and Readout
- For chemiluminescence detection, add the appropriate amount of chemilluminescent substrate to membrane and incubate for the recommended amount of time.
- For chemiluminescence detection, expose membrane to light-sensitive film or capture light signals with a CCD detection system to bands of target proteins or antigens. For fluorescence detection, image the membrane on a fluorescence imaging system using the appropriate excitation/emission settings.