Anti-BMX (phospho Y566)抗体(ab59409)

Bitte entschuldigen Sie die Verzögerung.

Ich habe nun aus dem Labor die Protokolle sowohl für die Behandlung der HeLa Zellen als auch das WB Protokoll für ab59409 bekommen.

Ich kopiere ihnen beide Protokolle unten in die Email.

Bitte lassen Sie mich wissen ob die Protokolle geholfen haben.

The protocol of sample treatment for Western blot (for ab59409) is following:

Grow Hela cells to ˜80% confluence in DMEM with 10% FBS at 37C and 5% CO2

Remove the culture medium and wash with DMEM without FBS

Starve the cells in DMEM with 0.2% FBS for 72 hours at 37C and 5% CO2 with changes starving medium (DMEM with 0.2% FBS) once a day (total three times)

After 72 hours, remove the medium, culture the cells in DMEM medium with 20% FBS (fresh) for 15 minutes at 37C and 5% CO2

Remove the medium and rinse the cells with 1x PBS buffer two times

Lyse the cells in lysis buffer and measure protein concentration

Run 10% SDS-PAGE with 50 ug per lane for 150V (for mini-gel)

Transfer to NC paper for Western blot





Blocking

1. Block membrane by incubating 1 hour at room temperature or overnight at 4℃ with shaking in Blocking Solution (5% BSA, 0.05% Tween-20 in TBS (50mM Tris, 100mM NaCl, pH 7.6).

Incubation with Primary Antibody

2. Dilute primary antibody at the appropriate dilution in Blocking Solution.

3. Incubate the membrane with diluted primary antibody overnight at 4℃ with agitation.

4. Remove antibody solution. Wash the membrane 3 times for 5-10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking. Note: Increase the concentration of Tween-20 to 0.1% reduces the background and increases the specificity, but it will reduce the sensitivity.

Incubation with Second Antibody

5. Incubate membrane with secondary AP conjugate diluted (according to manufacturer’s instructions) in Blocking Solution for 1 hour at room temperature with shaking.

6. Repeat Step 4.

7. Wash membrane with TBS for 2-5 minutes before proceeding Chemiluminescent Reaction.

Chemiluminescent Reaction

8. Prepare and use the Chemiluminescent substrate according to the manufacturer’s instructions.

9. Immediately wrap the membrane and expose to X-ray films for 10 second to 1 hour period. The exposure time may vary according to the amount of antibody and antigen.