Anti-ARMS2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ARMS2 on a Western blot?

ARMS2 is a 107-amino acid protein with a predicted molecular weight of approximately 12 kDa. On reducing SDS-PAGE you should see the primary band in the 12-14 kDa range. Because the protein is small, use a 12-15% gel or a gradient gel (4-20%) and confirm transfer to a 0.2 µm PVDF or nitrocellulose membrane — proteins under 15 kDa pass through 0.45 µm membranes readily. Endogenous expression is reported to be low in many tissues, so loading 30-50 µg of total protein and extended ECL exposure may be needed.

What is a good starting dilution for ARMSP-2 in Western blot?

Start at 1:1000 in 5% non-fat milk or BSA in TBST, with overnight incubation at 4°C. Because endogenous ARMS2 levels are typically low, you may need to titre between 1:500 and 1:2000 against your specific lysate. If background is high, switch the blocking buffer to 5% BSA and reduce primary to 1:2000. For IHC and IF, the antibody is brand-validated but optimal working dilutions should be determined empirically on your fixation and antigen retrieval conditions.

Does this antibody detect the A69S (rs10490924) variant of ARMS2?

The A69S polymorphism is a single residue substitution and is not expected to disrupt epitope recognition for a polyclonal raised against the full-length or near full-length protein, so both wild-type and A69S variants should be detected with comparable sensitivity. However, the antibody does not discriminate between the two alleles on Western blot — genotype must be confirmed by sequencing or allele-specific PCR. Note that the indel variant in linkage disequilibrium with HTRA1 destabilizes ARMS2 mRNA, so homozygous indel carriers may show markedly reduced or absent ARMS2 protein.

What positive and negative controls should I use for ARMS2 Western blot?

There is no widely accepted endogenous high-expresser cell line for ARMS2, which complicates positive controls. Many groups use transient overexpression of recombinant ARMS2 (FLAG- or HA-tagged) in HEK293 or COS-7 cells as a positive control, run alongside untransfected lysate as a negative control. Human retinal or RPE lysate can be used for endogenous detection but signal is weak. Including a siRNA knockdown or CRISPR knockout lysate is the most rigorous specificity control given the limited functional characterization of this protein.

Will this antibody cross-react with mouse or rat ARMS2?

Reactivity is validated for human only. ARMS2 is notable for being primate-specific or near primate-specific — orthologs in mouse and rat are either absent or highly divergent, depending on the annotation source. We do not recommend this antibody for rodent samples, and a negative result in mouse or rat tissue should not be interpreted as a technical failure. For comparative studies, use human cell lines, primate tissue, or transgenic mouse models expressing human ARMS2.

Why am I seeing multiple bands or no band at all with ARMS2?

Two common issues: first, ARMS2 expression is low in most tissues outside the retina, so absent signal often reflects biology rather than antibody failure — try overexpression lysate to confirm the antibody works in your hands. Second, ARMS2 has been reported to be unstable and rapidly degraded, particularly in indel-variant carriers, so additional lower-MW bands may represent degradation products. Use fresh lysates prepared with protease inhibitors, keep samples cold, and avoid repeated freeze-thaw of lysates. Bands above 20 kDa are unlikely to be ARMS2 and may indicate non-specific binding.

What sample preparation works best for detecting endogenous ARMS2?

ARMS2 is cytoplasmic, so standard RIPA or NP-40 lysis buffers are appropriate. Include a complete protease inhibitor cocktail given the protein's reported instability. For retinal or RPE tissue, homogenize quickly on ice and proceed directly to denaturation in Laemmli buffer at 95°C for 5 minutes. Avoid boiling for extended periods. Because the target is small (~12 kDa), do not over-run your gel — stop when the dye front is near the bottom but still on the gel to retain ARMS2 resolution.

How should I store the ARMSP-2 antibody?

Store at -20°C for long-term storage in the supplied buffer. Upon first thaw, aliquot into single-use volumes (5-10 µL) to avoid repeated freeze-thaw cycles, which degrade polyclonal performance. Working aliquots can be held at 4°C for up to two weeks. Do not store at -80°C unless specifically required by your institutional protocol, as cycling between -80°C and bench temperature is more damaging than -20°C storage. The 100 µg pack at 1:1000 dilution supports roughly 50-100 mini-blots depending on incubation volume.