Free Blocking Buffer*
Rockland's Affinity Purified anti-HA epitope tag polyclonal antibody detects HA tagged recombinant proteins by IHC on formalin fixed paraffin embedded tissue. Arrowheads point to expression of HA tagged proteins in endothelial cells of mouse aorta. Sections of 4 µm were prepared from representative paraffin blocks. Sections were then deparaffinized and rehydrated with xylene and alcohol. Citrate buffer antigen retrieval was performed for 30 min in a boiling jar. Anti-HA was diluted in blocking buffer at 1:2,000 and reacted at 4° C overnight followed by signal detection using horseradish peroxidase with DAB as the chromogenic substrate. Tissue was counterstained with Mayer's hematoxylin. Personal Communication, Behzad Yeganeh,U. Manitoba, Winnipeg, Canada.
Anti-HA epitope tag polyclonal antibody detects HA-tagged recombinant proteins by western blot. Polyclonal Rabbit anti-HA epitope tag, at a 1:2,000 dilution, was used to detect 1.0 µg of 12-Epitope Tag Protein Marker Lysate (p/n MB-301-0100) containing the HA epitope tag. A 4-20% gradient gel was used to resolve the protein by SDS-PAGE. The lysate was transferred to nitrocellulose using standard methods. After blocking, the membrane was probed with Rockland's anti-HA tag antibody for 1 h at room temperature followed by washes and reaction with a 1:20,000 dilution of IRDye® 800 conjugated Gt-a-Rabbit IgG (H&L) MX10 (code 611-132-122) for 30 min at room temperature. LICOR's Odyssey® Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.
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