Anti-EBV Bam HI Z antibody [BDI506] (ab21134)

I am planning western blotting of EBV Bam HI Z. I am interested in this antibody, but I cannot find any papers using this antibody for western blotting. If you know anything about it, please let me know.

ANSWER:

Thank you for your enquiry. I was not able to find any published references for this clone but I have the following information:

Positive controls are P3HR-1 and B95-8 cells induced with Na-butyrate (3mM) or Na-butyrate (3mM) and TPA (20ng/ml).

If the cells express spontaneously early antigens in at least 5% of the cells, MAb to EBV Bam HI-Z, ab21134, will detect the Zeta protein without induction on the blots.

The Zeta protein has also been detected in Raji cells induced with Na-butyrate (3mM) and TPA (20ng/ml).

Following is the Wetern blot protocol used by the lab.

Preparation for whole cell lysates

Cells are washed in PBS, scraping in PBS if necessary, and harvested by centrifugation. Cell pellets are solubilized directly in 1X SDS Loading buffer (63 mM Tris-HCl, pH 6.8, 2% SDS, 0.0025% Bromphenol Blue, 10% glycerol, 50 mM DTT) at 1x10^7 cells per ml. The extracts are heated in boiling water bath for 5 minutes and electrophoresed on a 10% Tris-Glycine SDS-PAGE gel. Lysate from approximately 1.5x10^5 cells (~15 µl) are loaded per lane. Transfer the electrophoresed proteins to a PVDF membrane and incubate the membrane for 30 minutes at room temperature in blocking solution (2% milk in PBS, 0.05% Tween-20). Incubate the membrane for 1 hour at room temperature in antibody diluent (1% milk in PBS, 0.05% Tween-20) containing 1-2ug/ml of anti-EBV antibody. Each laboratory should determine an optimum working titer for use in its particular application.

Wash the membrane at room temperature for 15 minutes with 3 changes of blocking solution. Incubate the membrane at room temperature for 1 hour in antibody solution containing a 1:10,000 dilution of anti-mouse conjugated to horseradish peroxidase. Each laboratory should determine an optimum working titer for use in its particular application.

Wash the membrane for 15 minutes with 3 changes of PBS. Develop peroxidase reaction, preferably with ECL+.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I am not seeing any bands of the correct size. I am using an EBV induced cell line. Can you tell me how the antibody was tested and what samples were used?

ANSWER:

Thank you for your enquiry and your patience.

Positive controls are P3HR-1 and B95-8 cells induced with Na-butyrate (3mM) or Na-butyrate (3mM) and TPA (20ng/ml). If the cells express spontaneously early antigens in at least 5% of the cells, MAb to EBV Bam HI-Z, ab21134, will detect the Zeta protein without induction on the blots. The Zeta protein has also been detected in Raji cells induced with Na-butyrate (3mM) and TPA (20ng/ml).

Following is the Wetern blot protocol used by the lab.

Preparation for whole cell lysates

Cells are washed in PBS, scraping in PBS if necessary, and harvested by centrifugation. Cell pellets are solubilized directly in 1X SDS Loading buffer (63 mM Tris-HCl, pH 6.8, 2% SDS, 0.0025% Bromphenol Blue, 10% glycerol, 50 mM DTT) at 1x10^7 cells per ml. The extracts are heated in boiling water bath for 5 minutes and electrophoresed on a 10% Tris-Glycine SDS-PAGE gel. Lysate from approximately 1.5x10^5 cells (~15 µl) are loaded per lane. Transfer the electrophoresed proteins to a PVDF membrane and incubate the membrane for 30 minutes at room temperature in blocking solution (2% milk in PBS, 0.05% Tween-20). Incubate the membrane for 1 hour at room temperature in antibody diluent (1% milk in PBS, 0.05% Tween-20) containing 1-2ug/ml of anti-EBV antibody. Each laboratory should determine an optimum working titer for use in its particular application.

Wash the membrane at room temperature for 15 minutes with 3 changes of blocking solution. Incubate the membrane at room temperature for 1 hour in antibody solution containing a 1:10,000 dilution of anti-mouse conjugated to horseradish peroxidase. Each laboratory should determine an optimum working titer for use in its particular application.

Wash the membrane for 15 minutes with 3 changes of PBS. Develop peroxidase reaction using colorimetric reagents.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.