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Protocol

Western Blotting

Western Blotting Solutions and Reagents

1.    10X Phosphate Buffered Saline (PBS): To prepare 1 L of 10X PBS buffer: 80g NaCl, 2g KCl, 36.3g
        Na2HPO4•12H2O, 2.4g KH2PO4, adjust pH to 7.4.
2.    1X Cell Lysis Buffer with protease/phosphatase inhibitors
3.    5X SDS Sample Buffer:1M Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 500mM DTT, 0.05% Bromophenol
       Blue
4.    Transfer Buffer:25 mMTris base, 0.2 M glycine, 20% (or 10%)methanol (pH 8.5)
5.    10X Tris Buffered Saline (TBS):To prepare 1 L of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6
       with HCl (use at 1X)
6.    Blocking Buffer:5% nonfat milk, 1X TBS, 0.1% Tween-20
7.    Wash Buffer/TBST:1X TBS, 0.1% Tween-20(100%)
8.    HRP-conjugate secondary antibody
9.    Chemiluminesence reagents

Cell Lysis Protocol

Westerns are performed using cell lysates from harvested cells.
1.    Treat cells by specific regulator for desired time, wash cells with 1X PBS.
2.    Detach adherent cells using a cell scraper or centrifuge suspended cells, and resuspend in 1X Cell 
       Lysis Buffer with inhibitors.
3.    Remove a small volume (50 μl). To perform a protein assay, determine the protein concentration for each
       cell lysate.
4.    To the remaining volume of cell lysate, add half volume of 5X SDS Sample Buffer.
5.    Boil each cell lysate at 100 °C for 5 min.
6.    Centrifuge at 14,000 rpm in a microcentrifuge for 5 min.
7.    Load equal amounts (30–45μg) cell lysate onto SDS-PAGE gels using loading tips, along with molecular
       weight markers.
8.    Run the gel and transfer to nitrocellulose for western immunoblotting.
 
Western Blotting Protocol

Membrane Blocking

1.    Block membrane by incubating 1 hour at room temperature or overnight at 4 °C with shaking in Blocking
       Solution(5% nonfat milk, 1X TBS, 0.1% Tween-20).

Incubation with Primary Antibody

1.    Dilute primary antibody at the appropriate dilution in Blocking Solution.
2.    Incubate the membrane with diluted primary antibody for 2 hours at 37 °C, or overnight at 4 °C with
       agitation.
3.    Remove antibody solution. Wash the membrane 3 times for 5 minutes each time at room temperature in
       TBST with shaking.

Incubation with Secondary Antibody

1.    Incubate membrane with diluted HRP-conjugate secondary antibody (according to manufacturer’s
       instructions) in Blocking Solution for 1 hour at room temperature with shaking.
2.    Remove antibody solution. Wash the membrane 3 times for 5 minutes each time at room temperature in
       TBST with shaking.


Chemiluminescent Reaction

1.    Prepare and use the Chemiluminescent substrate according to the manufacturer’s instructions.
2.    Chemiluminescent substrate lay evenly over the membrane. Put them into chemiluminescent imager for
       exposure.


Peptide Competition Protocol

Before proceeding Western Immunoblotting, add specific Blocking Peptide (Refer to blocking peptide catalog#) to the diluted primary antibody in a molar ratio of 10:1 (peptide to antibody) and incubate the mixture at 4℃ for overnight or at 37 °C for 2 hours.

Phosphatase experiment Protocol

After blocking membrane,wash membrane with TBST for 1-2 min, then add phosphatase to the diluted primary antibodywith the appropriate concentrationand incubate the mixture at 4℃ for overnight or at 37 °C for 2 hours.

 

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