Direct-Blot™ Western Blotting Protocol

 

Reagent List

1X Cell Lysis Buffer: 20mM Tris-HCl, pH 7.5, 150mM NaCl, 1% NP-40,2 mM EDTA, 1µg/ml leupeptin, 1µg/ml aprotinin ,1mM Na3PO4, 1mM PMSF, 5mM NaF, 3mM Na4P2O4 5X SDS Sample Buffer: 312.5mM Tris-HCl (pH 6.8) , 10% SDS (w/v), 250mM DTT, 50% Glycerol, 0.05% Bromophenol Blue (w/v) Use at 1X, 80.0g NaCl, 4.4g Na2HPO4, 2.4g KH2PO4, 2.0g KCl. Add ddH2O up to 10L, pH to 7.2 with HCl 10X SDS Running Buffer: Dissolve 144g of Glycine, 30g of Tris base and 10g SDS in 800ml of distilled H2O. Add distilled H2O to 1 liter. Use at 1X Transfer Buffer: 3.0g Tris base, 14.4g Glycine 200ml Methanol. Add distilled water to 1.0L

10X TBS-T (Tris-buffered saline containing Tween-20): Dissolve 80g of NaCl, 2g of KCl, 30g of Tris base and 10ml, Tween-20 in 800ml of distilled H2O. Adjust the pH to 7.4 with HCl. Add distilled H2O to 1 liter. Use at 1X (containing 0.1% Tween-20). Blocking Buffer: 1X TBS-T with 5% nonfat dry milk Wash Buffer: 1X TBS-T Direct-Blot™ Antibody Dilution Buffer: 1X TBS-T with 5% nonfat dry milk. **If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk. Alternate Blocking Buffer: 1X TBS-T with 4% Bovine Serum Albumin (BSA) Alternate Direct-Blot™ Antibody Dilution Buffer: 1X TBS-T with 4% Bovine Serum Albumin (BSA) Blotting Membrane: Nitrocellulose or PVDF membrane

 

Protocol Steps

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Sample Preparation:

 

1. Place cells in a microcentrifuge tube and centrifuge to collect the cell pellet.  


2. Lyse the cell pellet with 100µl of lysis buffer on ice for 30 min (For 1 X 10⁶ cells, lyse with 100µl of lysis buffer).


3. Centrifuge at 14,000 rpm (16,000xg) for 10 minutes at 4°C.


4. Transfer the supernatant to a new tube and discard the pellet. Remove 20µl of supernatant and mix with 20 µl of 2x sample buffer.  


5. Boil for 5 min. Cool at room temperature for 5 minutes. Microcentrifuge for 5 minutes.  


6. Load up to 40µl of sample to each well of a 1.5mm thick gel.
   Note: Guidelines for choosing gel percentages are based on protein size to be detected: 4-5% gel, >200 kD; 7.5% gel, 120-200 kD; 8-10% gel, 40-120 kD; 13% gel, 15-40 kD; 15% gel, < 20 kD.  


7. Set gel running conditions according to the manufacturer’s instructions. Transfer the proteins to a nitrocellulose or PVDF membrane with variable power settings according to the manufacturer’s instructions.  

 

Membrane Blocking:

 

8. Remove the blotted membrane from the transfer apparatus and immediately place in blocking buffer consisting of 5% nonfat dry milk/TBS-T.
   Note: If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk. 


9. Incubate the blot for 1 hour at room temperature, or overnight at 4°C with agitation. 

 

Antibody Incubation:

 

10. Dilute the Direct-Blot™ antibody to the recommended concentration/dilution in 5% nonfat dry milk/TBS-T (usually at a 1:1000-1:2000 dilution). Place the membrane in the Direct-Blot™ antibody solution and incubate for 2 hours at room temperature, or overnight at 4°C with agitation.
    Note: If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk.


11. Wash three times for 5 minutes each with Wash Buffer (TBS containing 0.1% Tween-20)  

 

Protein Detection:

 

12. Incubate membrane (protein side up) with 10ml of ECL (enhanced chemiluminescence substrate) for 1-2 minutes. The final volume required is 0.125ml/cm².


13. Drain off the excess detection reagent, wrap up the blots, and gently smooth out any air bubbles.  


14. Place the wrapped blots, protein side up, in an X-ray film cassette and expose to x-ray film. Exposures can vary from 5 seconds to 60 minutes.