Western Blot with SDS-PAGE
Introduction
Materials
- 2X SDS-PAGE Sample Buffer (adjust to pH 6.8, store at -20°C)
- 1.5g Tris base
- 4g SDS
- 20mL Glycerol
- 2mL Beta-Mercaptoethanol
- 0.02g Bromophenol blue
- 100mL ddH2O
- SDS-PAGE Running Buffer (store at 4°C, warm before use)
- 3.03g Tris base
- 24g Glycine
- 1.0g SDS
- 1000mL ddH2O
- Transfer Buffer (store at 4°C)
- 3.03g Tris base
- 14.4g Glycine
- 200mL Methanol
- 800mL ddH2O
- Blocking Buffer (store at 4°C)
- 100mL PBS Buffer
- 5g Non-fat dry milk
- Washing Buffer (store at 4°C)
- 0.5mL Tween 20
- 1000mL PBS Buffer
- PBS Buffer (adjust to pH 7.4, store at 4°C)
- 8.5g NaCl
- 1.4g Na2HPO4
- 0.2g NaH2PO4
- 1000mL ddH2O
- Primary Antibody Dilution Buffer (Adjust to pH 7.4, store at 4°C)
- 1g BSA
- 100mL Washing Buffer
- ECL kit
Procedure
SDS-PAGE
- Add 2X SDS-PAGE Sample Buffer to protein samples (1:1) and heat to 100°C for 5 minutes.
CRITICALDon’t boil too long to avoid proteins getting destroyed.
- Wash a gel with SDS-PAGE Running Buffer and load samples into the gel.
Samples containing multiple proteins require 10-60µg of protein per well. Purified target protein requires 0.05-1µg.
- Run the gel at 100V through the stacking part of the gel and turn the volts up to 200V after the proteins have gone through the stack and are migrating through the resolving gel.
- Allow migration to continue until the blue dye front is at the end of the glass plates, but has not migrated off the gel.
Transferring
- Soak a PVDF membrane in methanol for 30 seconds, and then place it in distilled water.
- Place the PVDF membrane, two fiber pads, and four Whatman papers (pre-cut) in a shallow tray filled with Transfer Buffer for a few minutes.
- Disassemble gel apparatus and carefully pry plates apart. Then cut off stacking gel with a clean razor blade and soak gel in Transfer Buffer for a few minutes.
- Open transfer apparatus gel cassettes with the black panel lying flat on the bottom of the tray filled with Transfer Buffer, the clear panel should be against the side of the tray.
- Prepare the transfer sandwich on the black panel in the tray filled with Transfer Buffer:
one fiber pad
two Whatman papers
SDS gel
PVDF membrane
two Whatman papers
one fiber pad
Note: Remove air bubbles by rolling a glass tube on the membrane.
- Cover the sandwich with the clear panel, fasten with the latch, and insert the gel cassette into the electrode module with the black panel facing the black cathode electrode panel.
- Insert the bio-ice cooling unit into the buffer chamber, and fill the buffer chamber with Transfer Buffer. Transfer for 1-2 hours at 4°C, stirring at a constant current of 100V.
- (optional) Stain with 1x Pongee S for one minute and destain in ddH2O and rinse it with PBS Buffer.
Blocking and Incubating
- Incubate the membrane with Blocking Buffer on a shaker for 1-2 hours at 37°C or overnight at 4°C.
- Dilute primary antibody with Primary Antibody Dilution Buffer and incubate the membrane with the diluted primary antibody on a shaker for 1 hour at 37°C or overnight at 4°C.
- Wash the membrane four times with Washing Buffer on the Shaker for 5-10 minutes each time.
Note: For HRP conjugated primary antibody, get rid of step 4 and step 5.
- Dilute secondary antibody with Blocking Buffer and incubate the membrane with the diluted secondary antibody on a shaker for 1 hour at 37°C or overnight at 4°C.
- Wash the membrane four times with Washing Buffer on the Shaker for 5-10 minutes each time.
Detection
- For HRP conjugated secondary antibody:
Detect protein with ECL kit (2mL/membrane). In a separate tube, mix black and white ECL solutions in a 1:1 ratio.
Aliquot solution onto membranes and wait for 1 min. Drain the ECL, wrap in plastic and expose to film.
Expositon time to the blots for 10 seconds, 1 minute, 5 minutes, and 20 minutes to visualize the chemiluminescence signal corresponds to the specific antibody-antigen reaction. Optimal dilutions of HRP conjugated secondary antibody should also be tested. Suggested starting dilutions to test are at the range of 1:5000 ~ 1:10,000.
- For IRDye 800 conjugated secondary antibody:
Rinse the membrane with PBS Buffer and develop the signal with Odyssey Infrared Imaging System according to its accompanying manual.
Optimal dilutions of dye-conjugated secondary antibodies should also be tested. Suggested starting dilutions to test are 1:5000, 1:10,000, and 1:20,000.
Comments
Western blots are used to detect specific proteins in a tissue sample or extract. Gel electrophoresis is applied to separate proteins by structure or length. The proteins are transferred to a membrane and then stained with antibodies specific to the target protein.