Anti-TIMP-4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for TIMP-4 on Western blot?

TIMP-4 migrates at approximately 22-24 kDa on reducing SDS-PAGE, corresponding to the mature secreted form of the protein after signal peptide cleavage. The full-length precursor is 224 amino acids, but the 26-residue signal peptide is removed during secretion. You may occasionally observe higher molecular weight bands around 50 kDa representing TIMP-4 in complex with pro-MMPs, particularly pro-MMP-2, though these complexes are often dissociated under standard denaturing conditions. If working with conditioned media or serum samples, concentrate your sample to improve detection since TIMP-4 is secreted at relatively low levels compared to TIMP-1 or TIMP-2.

Which tissue lysates serve as good positive controls for TIMP-4 expression?

Heart tissue provides the most reliable positive control for TIMP-4, as cardiac myocytes express relatively high levels compared to other tissues. Human brain, ovary, and skeletal muscle lysates also work, though expression levels are more variable. Colon tissue shows moderate expression. Avoid using liver, kidney, or lung as positive controls since TIMP-4 expression in these tissues is minimal. For cell lysates, try human cardiac fibroblasts or cardiomyocytes if available. Many transformed cell lines express little to no TIMP-4 under basal conditions, so you may need to collect conditioned medium rather than whole-cell lysate to detect secreted TIMP-4. Include a recombinant TIMP-4 standard if tissue access is limited.

What starting dilution should I use for TIMP-4 Western blot?

Start at 1:1000 dilution for Western blot, as recommended for this polyclonal antibody. This dilution typically yields clean signal with low background when using 20-30 µg total protein per lane and standard ECL detection. If your sample has low TIMP-4 expression or you are probing conditioned media, you may improve sensitivity by using 1:500 or concentrating your sample by ultrafiltration or TCA precipitation. Conversely, if you observe high background with abundant target, test 1:2000 or 1:3000. Blocking in five percent non-fat dry milk in TBST for one hour at room temperature works well for this target. Optimize exposure time rather than antibody concentration first.

Does this TIMP-4 antibody cross-react with other TIMP family members?

TIMP family members share structural homology, particularly in their N-terminal inhibitory domains, so cross-reactivity is a valid concern. This antibody is validated for human TIMP-4 and should be specific within the TIMP family, but we recommend running parallel lanes with recombinant TIMP-1, TIMP-2, and TIMP-3 if you need absolute confirmation of specificity. TIMP-4 migrates at a distinct size (22-24 kDa) compared to TIMP-1 (28 kDa) and TIMP-2 (21 kDa), which aids identification. If you detect multiple bands in your lysate, consider siRNA knockdown or knockout cell lines to confirm which band represents genuine TIMP-4 versus potential cross-reactive species or degradation products.

Will this rabbit polyclonal TIMP-4 antibody detect mouse or rat TIMP-4?

This antibody is validated for human TIMP-4. Mouse and rat TIMP-4 share approximately 75-80 percent sequence identity with human TIMP-4, so cross-reactivity is possible but not guaranteed. If you are working with mouse or rat samples, treat this as predicted rather than validated reactivity and include appropriate controls. Test the antibody at 1:500 to 1:1000 dilution and run a positive control lysate from mouse heart or brain alongside recombinant mouse TIMP-4 if available. Some rabbit polyclonal antibodies raised against human epitopes recognize rodent orthologs well, others do not. If signal is weak or absent, consider a species-specific antibody rather than troubleshooting further.

How should I store this TIMP-4 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. Once thawed, an aliquot can be kept at 4°C for up to one month if sodium azide or another preservative is present; check the product datasheet for formulation details. Do not store diluted antibody; prepare working dilutions fresh for each experiment. The 100 µg pack size should provide roughly 100-200 Western blots at 1:1000 dilution depending on your blotting volume. Polyclonal antibodies typically remain stable for two to three years at -20°C when stored properly. If you observe increased background or loss of signal over time, the antibody may have degraded.

Can I use this antibody for immunohistochemistry on TIMP-4 in human tissue?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry, and this rabbit polyclonal should work on formalin-fixed paraffin-embedded human tissue sections. TIMP-4 localizes to extracellular matrix and cytoplasm of expressing cells, with strongest staining in cardiac myocytes, neurons, and smooth muscle. Antigen retrieval is typically required; try citrate buffer pH 6.0 with heat-induced epitope retrieval first. Start with a 1:50 to 1:200 dilution and optimize based on signal intensity and background. Include heart or brain tissue as a positive control and TIMP-4-negative tissue such as liver as a negative control. Secreted proteins like TIMP-4 may show diffuse extracellular staining, which is expected.

What are common reasons for not detecting TIMP-4 in my Western blot?

First, confirm your sample expresses TIMP-4; many cell lines and tissues have low or undetectable levels. TIMP-4 is a secreted protein, so check conditioned medium rather than only whole-cell lysate. If working with tissue, use heart or brain lysate as a positive control. Second, verify you loaded sufficient protein; 30-50 µg per lane is often needed for endogenous TIMP-4 detection. Third, ensure your sample buffer is fresh and reducing agents have not oxidized. Fourth, consider that protease activity in your lysate may degrade TIMP-4 during extraction; add protease inhibitor cocktail and work quickly on ice. Finally, verify antibody storage and dilution, and try a shorter exposure if chemiluminescent signal is saturating quickly.