Anti-Serpin-A8 (Angiotensinogen) Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-A8 on a Western blot?

Serpin-A8 (angiotensinogen) migrates at approximately 52–65 kDa on reducing SDS-PAGE, depending on the extent of glycosylation. The unmodified polypeptide is 476 amino acids (~53 kDa), but the protein is heavily N-glycosylated in vivo, which shifts the apparent mass upward. Plasma-derived angiotensinogen typically runs closer to 60–65 kDa. Deglycosylation with PNGase F will collapse the band to the predicted 53 kDa core. If you observe multiple bands, consider proteolytic processing by renin or other proteases during sample handling.

What is the recommended starting dilution for Serpin-A8 antibody in Western blot?

We recommend starting at 1:1000 dilution for Western blot with this rabbit polyclonal. This dilution has been validated against human samples. Optimal dilution may vary with your detection system, substrate sensitivity, and target abundance. Hepatocyte lysates and serum or plasma samples typically yield strong signal at 1:1000 to 1:2000. If working with conditioned media or low-expression cell lines, begin at 1:500. Always include a titration series with your first batch of lysate to establish the linear range for your specific application.

Does this Serpin-A8 antibody cross-react with other serpin family members?

This antibody was raised against a region of human Serpin-A8 and has been validated for specificity against human angiotensinogen. Cross-reactivity with other serpin family members has not been systematically tested. Given the sequence divergence among serpin paralogs, significant cross-reactivity is unlikely, but we cannot rule it out without direct Western blot comparison using recombinant serpins or knockout lysates. If serpin specificity is critical for your experiment, consider including lysates from Serpin-A8 knockdown or knockout cells as negative controls to confirm on-target detection.

Will this antibody work with mouse or rat samples?

This antibody is validated for human Serpin-A8. Reactivity with mouse or rat angiotensinogen is predicted but not experimentally confirmed. Human and rodent angiotensinogen share approximately 65–70 percent sequence identity, concentrated in the structured core domain. Cross-reactivity depends on whether the immunogen region is conserved across species. If you plan to use mouse or rat samples, we recommend performing a pilot Western blot with positive control lysates (liver or plasma) at 1:500 to 1:1000 dilution. Contact us if you need species-specific validation data.

What positive control samples should I use for Serpin-A8 Western blots?

Human liver lysate is the most reliable positive control, as hepatocytes are the primary source of circulating angiotensinogen. Alternatively, use diluted human plasma or serum, which contains abundant Serpin-A8. HepG2 cell lysates also express detectable levels. Avoid using non-hepatic cell lines unless you have confirmed expression by qPCR or RNA-seq. For negative controls, consider Serpin-A8 siRNA-transfected hepatocytes or pre-incubating the antibody with blocking peptide if available. Renin-treated samples will show cleavage products and reduced full-length band intensity.

Can this antibody detect angiotensinogen in immunohistochemistry or immunofluorescence?

Triple Point Biologics antibodies are broadly validated for Western blot. This rabbit polyclonal should be compatible with IHC and IF, though optimal conditions will depend on your fixation and antigen retrieval protocol. For IHC on formalin-fixed paraffin-embedded tissue, start with heat-mediated antigen retrieval in citrate buffer, pH 6.0, and test dilutions from 1:100 to 1:500. Serpin-A8 is most abundant in hepatocytes; expect strong cytoplasmic and secretory staining. Titrate carefully to minimize background in non-hepatic tissues.

How should I store this Serpin-A8 antibody and what is the shelf life?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied in a glycerol-containing buffer that remains liquid at –20°C, so you may store as a single vial if you use it frequently. Under these conditions, the antibody remains stable for at least two years from the date of receipt. Avoid prolonged storage at 4°C, which can promote bacterial growth and proteolytic degradation. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer for each experiment.

Why am I seeing lower-molecular-weight bands below 53 kDa in my Serpin-A8 Western blot?

Lower-molecular-weight bands below the expected 52–65 kDa range usually indicate proteolytic cleavage. Angiotensinogen is a substrate for renin, which cleaves the N-terminal decapeptide to generate des-angiotensin-I angiotensinogen (des-AI-AGT), running ~1 kDa smaller. Additional proteases in plasma or lysates can produce further fragments. To minimize artifactual cleavage, work quickly on ice, add protease inhibitor cocktail immediately upon lysis or blood collection, and avoid multiple freeze-thaw cycles. If you are studying renin activity, these cleavage products may be biologically relevant readouts.