Anti-TIMP-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for TIMP-2 on a Western blot?

TIMP-2 migrates at approximately 21–24 kDa on reducing SDS-PAGE gels, corresponding to the 220-amino acid secreted protein. The predicted mass is around 24 kDa, but many laboratories observe a band closer to 21 kDa depending on glycosylation state and gel conditions. TIMP-2 contains three N-glycosylation consensus sites, though glycosylation is typically minimal. Under non-reducing conditions the protein may migrate slightly differently due to disulfide-bond stabilization. We recommend running both a positive-control lysate and recombinant TIMP-2 standard alongside your samples to confirm migration and specificity.

What is the recommended starting dilution for this TIMP-2 antibody in Western blot?

We recommend starting at 1:1,000 dilution in blocking buffer (five percent non-fat milk or BSA in TBS-Tween) for Western blot. This rabbit polyclonal has been validated at 1:1,000 against human cell lysates. Optimal dilution may vary depending on expression level in your tissue or cell line; if signal is weak, titrate down to 1:500 or 1:750. For abundant TIMP-2 sources such as conditioned medium from fibroblasts or endothelial cells, you may extend to 1:2,000. Always include a known TIMP-2-positive control to assess antibody performance and optimize signal-to-noise.

Does this TIMP-2 antibody cross-react with other TIMP family members?

TIMP-2 shares significant sequence homology with TIMP-1, TIMP-3, and TIMP-4, particularly in the N-terminal MMP-binding domain. This rabbit polyclonal was raised against a TIMP-2-specific immunogen; however, we have validated reactivity only with human TIMP-2 and have not formally tested cross-reactivity against other TIMP isoforms. If you are working in samples where multiple TIMPs are expressed, we recommend running lysates or recombinant proteins for TIMP-1, TIMP-3, and TIMP-4 alongside TIMP-2 to assess specificity in your system. Predicted cross-reactivity with mouse and rat TIMP-2 is high based on sequence homology.

Can I use this antibody for immunohistochemistry or immunofluorescence on tissue sections?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry; immunofluorescence protocols are also compatible with most of our rabbit polyclonals. For IHC on formalin-fixed paraffin-embedded tissue, antigen retrieval is typically required—citrate buffer at pH six, heated retrieval, is a common starting point for TIMP-2. TIMP-2 is a secreted protein, so you should expect diffuse extracellular and pericellular staining as well as cytoplasmic signal in producing cells. We recommend titrating the antibody from 1:100 to 1:500 for IHC and including a no-primary control to assess non-specific binding.

What positive control should I use to validate TIMP-2 detection?

Human fibroblasts, endothelial cells, and many carcinoma cell lines constitutively secrete TIMP-2, making conditioned medium or whole-cell lysates from these sources excellent positive controls. HT-1080 fibrosarcoma and HeLa cells are widely reported to express TIMP-2. Recombinant human TIMP-2 protein is also commercially available and serves as a definitive positive control at known concentrations. For tissue, normal kidney, liver, and heart express moderate to high levels of TIMP-2. If signal is absent in a presumed positive sample, verify sample preparation, confirm reducing conditions, and check that your transfer captured the 21–24 kDa range.

Will this antibody detect mouse or rat TIMP-2 in addition to human?

This antibody has been validated against human TIMP-2. Mouse and rat TIMP-2 share approximately 87–90 percent amino acid identity with human TIMP-2, so cross-reactivity is predicted but not formally validated. Many researchers have successfully used human TIMP-2 antibodies in mouse and rat samples, but we recommend confirming reactivity in your species by running a positive-control lysate from the target organism alongside recombinant human TIMP-2. If you observe no signal or multiple bands, the epitope recognized by this polyclonal may not be conserved. Titrate the antibody and include species-matched controls to assess performance.

How should I store the TIMP-2 antibody and what is the expected shelf life?

Store the antibody at –20 °C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is supplied in a stabilization buffer; aliquot upon receipt into volumes appropriate for your experiment, typically ten to twenty microliters per tube. Stored properly at –20 °C, rabbit polyclonal antibodies remain stable for at least two years. Avoid prolonged storage at four degrees, which can promote bacterial growth and gradual loss of activity. If you observe increased background or loss of signal over time, prepare a fresh aliquot and re-optimize dilution.

Does TIMP-2 form complexes with MMPs that affect antibody detection?

TIMP-2 binds tightly to the catalytic domain of multiple MMPs, forming stable one-to-one complexes. In biological samples—particularly conditioned medium, serum, or tissue extracts—a substantial fraction of TIMP-2 may be bound to pro-MMP-2, MMP-2, or MT1-MMP. Most polyclonal antibodies, including this one, recognize both free and MMP-complexed TIMP-2, so you should detect total TIMP-2 on a Western blot. However, complex formation can shift the apparent molecular weight slightly or alter epitope accessibility. If you need to distinguish free from bound TIMP-2, consider native PAGE, co-immunoprecipitation, or ELISA formats that discriminate complex state.