Anti-Paraplegin Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Paraplegin on Western blot?

Paraplegin (SPG7) migrates at approximately 88-90 kDa on reducing SDS-PAGE gels, which is consistent with the 795-amino acid polypeptide. You may observe additional bands around 80-85 kDa representing proteolytic fragments or processed forms, particularly in aged mitochondrial preparations. Because Paraplegin resides in the mitochondrial inner membrane, enrichment with mitochondrial fractionation will improve signal-to-noise ratio over whole-cell lysates. Always include a mitochondrial marker such as VDAC or COX IV to confirm fractionation quality and exclude cytosolic contamination.

What starting dilution should I use for Paraplegin Western blot?

We recommend starting at 1:1000 dilution for Western blot, which has been validated in-house using human cell lysates. Depending on your mitochondrial enrichment efficiency and protein loading, you may need to adjust between 1:500 and 1:2000. Paraplegin expression is relatively low in many cell lines; 20-40 µg of mitochondrial-enriched lysate typically yields clearer bands than 50 µg of whole-cell extract. Incubate overnight at 4°C for optimal signal. Standard rabbit polyclonal secondaries at 1:5000 to 1:10000 work well with chemiluminescent detection.

Does this Paraplegin antibody cross-react with mouse or rat samples?

This antibody is validated for human Paraplegin. Cross-reactivity with mouse and rat is predicted based on sequence homology—human SPG7 shares approximately 85-88 percent identity with mouse and rat orthologs across the immunogenic region. However, we have not formally validated reactivity in these species. If you are working with rodent samples, we suggest testing at the recommended 1:1000 dilution first and including a human positive control lysate in parallel. Mitochondrial preparations from mouse heart or liver are reasonable starting tissues given high Paraplegin expression in those organs.

What positive and negative controls should I use for Paraplegin experiments?

HeLa or HEK293T mitochondrial extracts serve as reliable positive controls due to constitutive Paraplegin expression. Skeletal muscle and heart tissue lysates also express Paraplegin abundantly and reflect physiologically relevant levels. For negative controls, consider using SPG7 knockout or siRNA-depleted cell lines if available, or cytosolic fractions that should lack mitochondrial inner membrane proteins. Blocking peptide controls are not included with this product. Running parallel blots with an anti-VDAC or anti-TOM20 antibody confirms mitochondrial enrichment and equal loading, which is critical given Paraplegin's low abundance in whole-cell lysates.

Can I use this antibody for immunofluorescence of Paraplegin in mitochondria?

Triple Point Biologics antibodies are broadly validated for immunofluorescence applications, and this rabbit polyclonal format is suitable for IF. Because Paraplegin localizes to the mitochondrial inner membrane, you will need to permeabilize cells thoroughly—0.2 to 0.5 percent Triton X-100 or digitonin-based permeabilization works better than saponin for inner-membrane antigens. Start with a 1:100 to 1:200 dilution and co-stain with MitoTracker or an outer membrane marker like TOM20 to confirm mitochondrial localization. Expect punctate or tubular staining that overlaps with mitochondrial networks. Methanol fixation may improve epitope accessibility compared to paraformaldehyde alone.

Why do I see multiple bands on my Paraplegin Western blot?

Multiple bands likely reflect proteolytic processing or degradation products intrinsic to Paraplegin biology. The m-AAA protease complex undergoes regulated cleavage, and Paraplegin itself can be a substrate for quality-control proteolysis within mitochondria. Bands below the expected 88-90 kDa may represent N- or C-terminal truncations, especially in stress conditions or prolonged sample handling. To minimize artifactual degradation, lyse cells quickly on ice with protease inhibitor cocktail, avoid freeze-thaw cycles, and load fresh or single-thawed mitochondrial preps. A dominant band at full-length MW with faint lower species is typical; diffuse smearing suggests sample degradation rather than biology.

How should I store this Paraplegin antibody for long-term stability?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase background. The product is supplied at 100 µg in a buffered solution; aliquot into 10-20 µL volumes immediately upon receipt if you plan intermittent use over months. For short-term use within four weeks, storage at 4°C is acceptable. Avoid prolonged exposure to room temperature and do not dilute stock solution for storage, as this accelerates degradation. Addition of 50 percent glycerol permits storage at -20°C without freezing, reducing mechanical stress on the antibody, though this is optional for polyclonal rabbit IgG.

Does Paraplegin expression vary across tissues and what loading should I expect?

Paraplegin is ubiquitously expressed but levels vary widely by tissue oxidative capacity. Cardiac and skeletal muscle, brain, and kidney show the highest expression due to dense mitochondrial content. Cultured cell lines typically express lower levels. For Western blot, load 20-40 µg of mitochondrial-enriched lysate or 50-80 µg of whole-cell lysate from high-expresser tissues. In low-expresser lines such as some lymphoblasts or fibroblasts, you may need 80-100 µg whole-cell protein or further mitochondrial enrichment. Longer exposures—up to five minutes for chemiluminescence—may be necessary. Validating equal mitochondrial loading with COX IV or citrate synthase antibodies is more informative than total protein stains.