Anti-Neuroserpin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Neuroserpin (SERPINI1) runs at approximately 55 kDa on SDS-PAGE under reducing conditions, though the predicted molecular weight from the 410-amino acid sequence is around 46 kDa. The discrepancy reflects post-translational modifications, particularly N-glycosylation at three consensus sites. Depending on glycosylation status and tissue source, you may observe bands between 50-60 kDa. Neuronal lysates typically show the fully glycosylated form. Treatment with PNGase F or other deglycosylation enzymes will shift the band closer to the predicted 46 kDa, confirming identity if needed.

What is the recommended starting dilution for this Neuroserpin antibody in Western blot?

We recommend starting at 1:1000 dilution in blocking buffer for Western blot, based on validation with human neuronal lysates. This rabbit polyclonal typically provides clear signal at this dilution when detecting endogenous Neuroserpin in brain tissue or neuronal cell lines. If signal is weak, you can increase concentration to 1:500, though higher backgrounds may appear. For recombinant protein or overexpression models, 1:2000 to 1:5000 often suffices. Always include a negative control lacking Neuroserpin to confirm band specificity, as serpins share structural homology.

Does this antibody cross-react with mouse and rat Neuroserpin?

This antibody shows validated reactivity with human Neuroserpin and predicted cross-reactivity with mouse and rat based on sequence homology. Human and mouse Neuroserpin share approximately 85 percent amino acid identity, with conservation highest in the serpin domain. Rat homology is similarly high. We have not formally validated performance in rodent samples, so if working with mouse or rat brain tissue, plan to optimize dilution and include positive controls such as cortex or hippocampal lysate known to express Neuroserpin. Primate samples (monkey) are also predicted to cross-react given high sequence conservation.

What positive control tissue or cell line should I use for Neuroserpin detection?

Human brain cortex or hippocampus lysates serve as strong positive controls, as Neuroserpin is highly expressed in neurons of these regions. For cell lines, differentiated neuronal models such as SH-SY5Y (after differentiation with retinoic acid) or primary cortical neuron cultures express detectable endogenous Neuroserpin. Recombinant human Neuroserpin protein is also suitable for validating antibody recognition and confirming expected molecular weight. Avoid using HeLa, HEK293, or other non-neuronal lines as negative controls unless you verify low or absent expression by qPCR, since basal serpin expression patterns vary.

Can this antibody detect Neuroserpin polymers associated with FENIB disease?

This polyclonal antibody recognizes native Neuroserpin and should detect polymeric forms under non-reducing or semi-denaturing conditions, as epitopes within residues targeted by polyclonals often remain accessible in polymers. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) results from mutations causing intracellular polymerization. To visualize polymers, run samples under non-reducing conditions or use native PAGE; high-molecular-weight species or smearing above the monomer band may appear. For inclusion body detection in tissue, immunohistochemistry or immunofluorescence on fixed brain sections is more informative than Western blot, and this antibody is validated for Western blot; IHC validation is in progress.

What sample preparation steps are critical for detecting secreted Neuroserpin?

Because Neuroserpin is secreted by neurons, you can detect it in both cell lysates and conditioned media. For lysates, use RIPA or similar buffer with protease inhibitors; avoid boiling samples excessively, as serpin structure can be sensitive to prolonged heat. For conditioned media, concentrate samples 10- to 20-fold using centrifugal filters (10 kDa cutoff) to improve detection, since secreted levels are often lower than intracellular pools. Include serum-free or low-serum culture conditions for at least 24 hours before collection to reduce albumin interference. Always include a loading control for media samples, such as Ponceau staining.

How should I store this Neuroserpin 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 100 µg pack size is suitable for dividing into 10-20 µL aliquots upon receipt. Rabbit polyclonal antibodies typically remain stable for at least two years at -20°C when stored properly. For short-term use (up to one month), you may keep a working aliquot at 4°C with 0.02 percent sodium azide as preservative. Avoid storing in frost-free freezers due to temperature cycling. If you observe precipitate after thawing, centrifuge briefly before use.

Does Neuroserpin require special blocking conditions to reduce background in Western blot?

Standard blocking with 5 percent non-fat dry milk in TBST for one hour at room temperature is usually sufficient for this rabbit polyclonal. However, if you observe high background, switch to 3-5 percent BSA in TBST, as milk proteins can sometimes cause non-specific binding with serpin antibodies. Ensure your neuronal lysates are fresh and protease inhibitors are included, since degraded samples increase background smearing. Extended blocking (overnight at 4°C) rarely improves signal-to-noise for this target. Including 0.05-0.1 percent Tween-20 in all wash steps helps reduce non-specific binding without compromising Neuroserpin detection.