Anti-BBS-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for BBS-1 on a Western blot?

BBS-1 migrates at approximately 65 kDa on SDS-PAGE, corresponding to the full-length 593-amino acid protein. Some researchers report bands slightly higher due to post-translational modifications or gel conditions. If you observe multiple bands, consider that BBS-1 can undergo phosphorylation. We recommend running a positive control lysate from ciliated cells such as hTERT-RPE1 or IMCD3 alongside your samples. Start with the 1:1000 dilution we validate, but titre if background is high or signal weak in your specific system.

Which cell types or tissues express BBS-1 at detectable levels?

BBS-1 is broadly expressed but enriched in ciliated cell types. Retinal pigment epithelium, kidney epithelial cells, olfactory neurons, and neural tissues show robust expression. For positive controls in Western blot or IHC, consider RPE1 cells after serum starvation to induce ciliogenesis, mouse kidney sections, or retinal lysates. Expression is generally low in non-ciliated or poorly ciliated lines like HeLa unless ciliation is chemically induced. BBSome components including BBS-1 are often below detection in whole-tissue homogenates without enrichment.

Is this antibody validated in mouse or other model organisms?

This antibody is validated in human samples. Cross-reactivity with mouse and rat BBS-1 is predicted based on sequence homology; human and mouse BBS-1 share approximately 89% identity across the full-length sequence. Several researchers have successfully used rabbit polyclonal BBS-1 antibodies in mouse tissue, but we recommend confirming specificity in your hands with a Bbs1 knockout or knockdown control if working outside validated human samples. Zebrafish or Drosophila cross-reactivity is less likely due to greater sequence divergence.

What dilution should I use for immunofluorescence of BBS-1 in cilia?

While this antibody is validated for Western blot at 1:1000, immunofluorescence typically requires optimization. Start at 1:200 to 1:500 for ciliary staining and titre based on signal-to-background in your system. Use ciliated cells such as serum-starved RPE1 or differentiated airway epithelial cultures. BBS-1 localizes to the ciliary membrane and base, so co-stain with a ciliary marker like acetylated tubulin or ARL13B to confirm specificity. Expect punctate signal along the cilium and at the basal body. Methanol fixation often preserves BBSome antigens better than paraformaldehyde alone.

Can this antibody distinguish between different BBS-1 isoforms or splice variants?

Human BBS1 has limited characterized splice variation; the predominant isoform is the canonical 593-amino acid sequence. Minor variants have been reported in transcript databases but their protein-level expression and functional relevance remain unclear. This polyclonal antibody is raised against a broad epitope region and will likely recognize canonical BBS-1 and any expressed variants containing that region. If you suspect variant expression in your model, consider running samples from BBS1 patient fibroblasts or engineered knockouts as negative controls to confirm band identity.

What controls should I include when using this antibody to study BBSome function?

Include a BBS1-depleted control using siRNA or CRISPR knockout to confirm specificity, particularly for immunofluorescence or co-immunoprecipitation experiments. Ciliated versus non-ciliated conditions serve as functional positives and negatives; serum starvation for 24-48 hours induces cilia in RPE1 or NIH3T3 cells. For pathway studies, consider that BBS-1 loss disrupts ciliary localization of other BBSome subunits and impairs sonic hedgehog signaling. Positive control lysates from wild-type ciliated cells and negative controls from patient-derived BBS1-mutant cells provide the cleanest validation.

How should I store this antibody and what is the expected shelf life?

Store the antibody at -20°C as supplied. Avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. For frequent use, aliquot upon receipt into single-use volumes and keep working aliquots at 4°C for up to one month. Polyclonal antibodies typically retain activity for two to three years when stored properly at -20°C, though activity may gradually decline. If you observe decreased signal over time, check for precipitation or cloudiness and consider re-titrating. Addition of glycerol or BSA as stabilizers is generally unnecessary for this format.

What sample preparation works best for detecting BBS-1 in Western blot?

Use standard RIPA or NP-40 lysis buffers supplemented with protease and phosphatase inhibitors, as BBS-1 can be phosphorylated. Load 20-30 µg total protein per lane for whole-cell lysates; ciliary or membrane-enriched fractions may require less. BBS-1 is a soluble peripheral membrane protein, so harsh detergents are not necessary. For ciliary studies, consider enriching cilia by shear-off or differential centrifugation to increase signal. Reducing conditions are standard; we have not observed disulfide-linked oligomers. Transfer to PVDF at 100V for 90 minutes or use semi-dry transfer for optimal recovery of this 65 kDa target.