重组Anti-HGF抗体[EPR12230] (ab178395)

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The western blot image shown on the datasheet for ab178395 was obtained using denaturing and reducing conditions.

I have copied below the WB protocols that have been used. Please note these would be a guideline only and may require individual optimization.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.




Cell Lysate preparation



Harvest 3.0*108 cells.
Add 3 ml 1×Hot lysis buffer .
Mix the cells and buffer well, then boil the tube for 15-30 min. (Take the tube out and mix them between whiles during incubation. )
Sonication. 3 seconds, intervals 3 seconds, 25-30 times (40kW) until the cell clumps all scattered and the liquid is clear.
Centrifugation. Centrifugate the tube at 10,000rpm at 4℃ for 10min and then put the supernatant at 4℃ temporarily.
Protein concentration quantitative analysis by BCA method.
Adjust concentration of the cell lysate to 4 mg/ml with 1×Hot lysis buffer.
Adjust to 2mg/ml protein with 2×sample buffer, keep 5min in boiling water. Then ready to use.





1×Hot lysis Buffer

10 mM Tris-Hcl(pH8.0)

1%SDS

1.0 mM Na-Orthovanadate

ddH2O



2×Sample Buffer

125mM Tris-Hcl(pH6.8)

2.5% SDS

0.04% Bromophenol Blue

25% Glycerol:

713mM ß-Mercaptoethanol:

ddH2O











Tissue Lysate preparation



Cut the frozen tissue into small piece with scissor. Grind the small piece into powder in mortar and transfer them to pre-cold centrifuge tube.



* The scissors, mortar and pestle should be pre-cold in -80 degree overnight.

Add 1×Hot lysis buffer, 2 times the volume of the tissue power.
Mix the tissue and buffer well, then incubate tube at 95-100°C for 15-30 min. (Take the tube out and mix them between whiles during incubation. )
Sonication. 3 seconds, intervals 3 seconds, 25-30 times (40kW) until the tissue clumps all scattered and the liquid is clear.
Centrifugation. Centrifugate the tube at 10,000rpm at 4℃ for 10min and then put the supernatant at 4℃ temporarily.
Measure the protein concentration by BCA method.
Adjust concentration of the tissue lysate to 4 mg/ml with 1×Hot lysis buffer.
Adjust to 2mg/ml protein with 2×sample buffer, keep 5min in boiling water. Then ready to use.





* 1×Hot lysis buffer:

10 mM Tris-Hcl(pH8.0)

1%SDS

1.0 mM Na-Orthovanadate

ddH2O





*2×Sample Buffer:

125mM Tris-Hcl(pH6.8)

2.5% SDS

0.04% Bromophenol Blue

25% Glycerol:

713mM ß- Mercaptoethanol:

ddH2O









Western blot protocol



1. Take the membrane out of 4℃ refrigerator. Treat the membrane with 99.5% methanol for 15 sec..

2. Rinse the membrane with deionized water for 5 min.

3. Block the membrane using 5% NFDM/TBST. Swing slightly for overnight at 4℃ on shaking table.

4. Wash the membrane with 1×TBST for 10 min.

5. Incubate the membrane with suitable dilution of primary antibody in 5% NFDM/TBST for 1h at RT on shaking table. Swing slightly.

6. Wash the membrane 3 times with 1×TBST, 10 min per time.

7. Incubate the membrane with 1:1000 dilution of the secondary antibody in 5% NFDM/TBST for 1h at RT on shaking table. Swing slightly.

*Secondary antibody information: Pierce, Goat Anti-Rabbit IgG, (H+L), Peroxidase Conjugated.

8. Wash the membrane with 1×TBST 3 times，10min per time.

9. Incubate the membrane with ECL substrate for 5min. at RT. (Mix reagent 1 and reagent 2 at the ratio of 1:1. Before using, place the mixture in dark place for 5min.)

10. Remove excess ECL substrate and sandwich the membrane in transparent plastic wrap.

11. Acquire image using darkroom development techniques. Expose the membrane for 3 min. (If the signal is very strong, re-expose the membrane for less minute).

When ab178395 was validated in flow, permeabilized A549 cells were used.