BBS-1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant BBS1 on SDS-PAGE and Western blot?

Recombinant BBS1 (REC-BBS1) spans the full 593-residue sequence of human BBS1 (UniProt Q8NFJ9), giving a predicted molecular weight of approximately 67 kDa. On denaturing SDS-PAGE, the band typically migrates at 68–72 kDa — a slight upward shift attributable to post-translational modifications retained from the HEK293 expression system. Purity is >90% by SDS-PAGE. If your Western blot shows a diffuse band or doublet near 70 kDa, this is consistent with phosphorylation or other PTMs and is not indicative of degradation.

Does recombinant BBS1 undergo any post-translational processing or exist as multiple isoforms?

The canonical human BBS1 sequence (UniProt Q8NFJ9, isoform 1) is the basis for REC-BBS1 — no truncation or alternative splicing is introduced. Because expression is in HEK293 mammalian cells, the protein carries human-relevant phosphorylation and glycosylation that a bacterial or insect-cell system would not reproduce. No known proteolytic cleavage event converts BBS1 to a shorter processed form under physiological conditions. The intact 593-residue protein is the biologically relevant assembly-competent form for BBSome co-complex studies.

What protein interactions can I study with recombinant BBS1 in pull-down or co-IP experiments?

REC-BBS1 is well-suited as bait or prey in pull-down assays interrogating BBSome assembly. Published work has used recombinant BBS1 to probe direct binding to RAB3IP/Rabin8 — the guanine nucleotide exchange factor for Rab8a — and to other BBSome subunits (BBS2, BBS7, BBS9). For pull-down, we recommend using REC-BBS1 at 1–5 µg per reaction in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — the native storage buffer — supplemented with 0.1% NP-40 to reduce non-specific binding. Confirm pulled complexes by Western blot using the matched antibody RP-BBS1.

What buffer conditions are optimal for recombinant BBS1 binding assays or biophysical characterisation?

REC-BBS1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a buffer chosen to maintain solubility and the native fold of the assembled scaffold domain. For SPR or ITC experiments, dialysing into a matched running buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween-20) at 4°C overnight is advisable. Avoid buffers below pH 6.5 or above pH 8.5, and keep glycerol at ≥5% if diluting significantly, as BBS1 has a tendency to aggregate at low ionic strength without a stabilising co-solute.

Is recombinant BBS1 an enzyme — does it have catalytic activity, and is there a standard activity assay?

BBS1 is a structural scaffold subunit of the BBSome coat complex, not a catalytic enzyme. It does not possess intrinsic ATPase, GTPase, kinase, or protease activity. Consequently, there is no standard small-molecule substrate activity assay for BBS1 in isolation. Functional characterisation is typically assessed by complex reconstitution (co-sedimentation or SEC-based assembly assays with other BBSome subunits) or by binding affinity measurements against partners such as RAB3IP/Rabin8. REC-BBS1 is therefore most appropriate as a positive control antigen, structural bait, or reference protein rather than an enzymatically active reagent.

How much recombinant BBS1 should I load as a positive control on a Western blot with the RP-BBS1 antibody?

For Western blot positive controls using the matched antibody RP-BBS1 (/anti-bbs-1-rabbit-polyclonal-antibody), load 10–50 ng of REC-BBS1 per lane. At 50 ng you should see a clean, strong band at approximately 68–72 kDa with standard ECL detection. If you are titrating alongside cell lysate, start at 20 ng to avoid overexposure relative to endogenous signal. RP-BBS1 is guaranteed compatible with REC-BBS1 for Western blot — both originate from the same TPB development pipeline — so band identity is unambiguous when run in parallel.

Can I use REC-BBS1 to validate the RP-BBS1 rabbit polyclonal antibody for Western blot or IHC?

Yes — this is one of the primary use cases for REC-BBS1. Because RP-BBS1 was raised and quality-controlled against the same full-length human BBS1 sequence represented by REC-BBS1, the recombinant is a reliable positive control antigen for Western blot validation. For antibody titration, run 25–100 ng REC-BBS1 alongside your lysate of interest and titrate RP-BBS1 from 1:500 to 1:5000. For IHC validation workflows requiring soluble antigen spiking or dot-blot confirmation, REC-BBS1 at 100–500 ng provides a clean signal at the expected molecular weight without background from endogenous proteins.

How should I store and handle recombinant BBS1 to maintain activity and avoid degradation?

REC-BBS1 is shipped on dry ice and should be stored at -20°C immediately upon receipt. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — stabilises the protein during freeze-thaw, but repeated cycling degrades both solubility and binding competence. Aliquot into single-use volumes before the first freeze. Working dilutions in assay buffer can be held at 4°C for up to 48 hours; do not leave the protein at room temperature for extended periods. Endotoxin is <0.1 EU/µg by LAL assay, so REC-BBS1 is suitable for cell-based experiments where LPS contamination would confound results.