Anti-Gastricsin Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for gastricsin on a Western blot?

Mature gastricsin migrates at approximately 42 kDa on reducing SDS-PAGE, corresponding to the 388-residue active enzyme after removal of the N-terminal propeptide. You may also detect pepsinogen C, the zymogen precursor, at approximately 45 kDa depending on your sample source and processing conditions. Gastric mucosal lysates often show both forms, while gastric juice typically contains predominantly the mature 42 kDa band. Because gastricsin is heavily glycosylated in some contexts, minor shifts in apparent molecular weight can occur. Always include a positive control lysate from gastric tissue to confirm the expected migration pattern.

What starting dilution should I use for Western blot with this gastricsin antibody?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this rabbit polyclonal antibody. Optimal dilution will depend on your sample type and gastricsin expression level. Gastric mucosa and pyloric gland lysates typically yield strong signals at 1:1000 to 1:2000, while serum or gastric juice samples may require 1:500 for detection. Perform a dilution series (1:500, 1:1000, 1:2000) with your specific sample to balance signal intensity and background. Use a high-quality secondary anti-rabbit IgG and standard chemiluminescent detection. Incubate primary antibody overnight at four degrees Celsius for best results.

Is this gastricsin antibody cross-reactive with pepsin A or other aspartic proteases?

Cross-reactivity with pepsin A (pepsin isoform 3A, encoded by PGA3, PGA4, PGA5) has not been formally validated but is unlikely given the sequence divergence between gastricsin and pepsin A isoforms, particularly in the antigenic regions. Gastricsin shares approximately 70 percent sequence identity with pepsin A, so some epitope overlap is theoretically possible. If you are working with complex gastric lysates containing multiple aspartic proteases, confirm specificity by including recombinant gastricsin and pepsin A controls on the same blot. Predicted cross-reactivity with cathepsin D or other aspartic proteases outside the pepsinogen family is low based on sequence homology.

What is the best positive control tissue or cell line for gastricsin immunodetection?

Human gastric mucosa is the gold-standard positive control, as mucous neck cells and pyloric glands synthesize pepsinogen C constitutively. Fresh-frozen or formalin-fixed paraffin-embedded gastric tissue sections work well for immunohistochemistry. For Western blot, prepare whole-cell lysates from human gastric corpus or antral biopsies. Gastric cancer cell lines such as MKN45 or AGS may express detectable gastricsin, but levels are variable and generally lower than normal mucosa. Gastric juice is another excellent positive control for Western blot, though it requires dialysis or concentration before loading. A recombinant gastricsin standard is ideal for verifying antibody specificity if tissue is unavailable.

Does this antibody detect gastricsin in mouse or rat stomach tissue?

This antibody is validated for human gastricsin. Reactivity with mouse or rat gastricsin is predicted but not experimentally confirmed. Mouse and rat pepsinogen C orthologs share approximately 75 to 80 percent sequence identity with human gastricsin, so cross-reactivity is plausible. If you plan to use rodent samples, perform a pilot experiment with mouse or rat gastric mucosa lysate alongside human positive control. Start at the recommended 1:1000 dilution and compare band patterns. Expect the mature protease to migrate near 42 kDa in rodents as well. Contact us if you need validation data for non-human species; we can discuss custom testing options.

How should I prepare gastric tissue lysates to preserve gastricsin for Western blot?

Gastricsin is an aspartic protease active at low pH, so avoid acidic lysis buffers that may trigger autocatalytic activity or substrate degradation. Use neutral pH lysis buffer (pH 7.2 to 7.6) containing protease inhibitors, including pepstatin A to block aspartic protease activity during extraction. Snap-freeze gastric tissue in liquid nitrogen immediately after collection and homogenize while still frozen. RIPA buffer with protease inhibitor cocktail works well for total protein extraction. Keep samples on ice throughout and store lysates at minus 80 degrees Celsius. Avoid repeated freeze-thaw cycles, which can degrade gastricsin and reduce signal intensity on blots.

Can this gastricsin antibody distinguish between the zymogen and mature active forms?

This polyclonal antibody recognizes epitopes present in both pepsinogen C (the zymogen) and mature gastricsin, so it does not selectively distinguish activation states by immunodetection alone. The two forms are best differentiated by molecular weight on Western blot: pepsinogen C migrates at approximately 45 kDa, while the mature enzyme runs at 42 kDa after propeptide cleavage. In gastric mucosa lysates, you will typically see both bands, reflecting intracellular zymogen and secreted or activated enzyme. To assess activation specifically, combine immunoblotting with enzymatic activity assays or use sample fractionation to separate intracellular (zymogen-enriched) from extracellular (mature enzyme-enriched) compartments.

What are the recommended storage conditions for this gastricsin antibody?

Store the antibody at minus 20 degrees Celsius as supplied in the original vial. For long-term storage beyond six months, minus 80 degrees Celsius is preferable to maintain titre. Avoid repeated freeze-thaw cycles, which can reduce antibody activity; aliquot into single-use volumes if you plan infrequent experiments. Once thawed, the antibody can be held at four degrees Celsius for up to one month with addition of sodium azide (0.02 to 0.05 percent final concentration) as preservative. Do not store diluted antibody in working buffer for more than one week. If you observe increased background or loss of signal over time, prepare a fresh aliquot from frozen stock.