Bovine 72 kDa type IV collagenase (MMP2) ELISA Kit
The Bovine 72 kDa Type IV Collagenase (MMP2) ELISA Kit is a valuable tool for accurate and reliable detection of MMP2 levels in bovine serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.MMP2, also known as gelatinase A, is an important enzyme involved in extracellular matrix degradation and tissue remodeling. It plays a critical role in processes such as wound healing, tissue repair, and tumor invasion.
By measuring MMP2 levels, researchers can gain insights into various physiological and pathological conditions, including cancer, cardiovascular diseases, and inflammatory disorders.With its robust performance and user-friendly design, the Bovine 72 kDa Type IV Collagenase (MMP2) ELISA Kit is a valuable asset for studying the role of MMP2 in different biological processes and diseases, paving the way for potential therapeutic interventions.
Product Name:
Bovine 72 kDa type IV collagenase (MMP2) ELISA Kit
PEX, the C-terminal non-catalytic fragment of MMP2, posseses anti-angiogenic and anti-tumor properties and inhibits cell migration and cell adhesion to FGF2 and vitronectin. Ligand for integrin alpha-v/beta-3 on the surface of blood vessels.
Uniprot:
Q9GLE5
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant bovine 72 kDa type IV collagenase
Sub Unit:
Interacts (via the C-terminal hemopexin-like domains-containing region) with the integrin alpha-V/beta-3; the interaction promotes vascular invasion in angiogenic vessels and melamoma cells. Interacts (via the C-terminal PEX domain) with TIMP2 (via the C-terminal); the interaction inhibits the degradation activity. Interacts with GSK3B.
Research Area:
Cancer
Subcellular Location:
Secreted Extracellular space Extracellular matrix Membrane Nucleus Colocalizes with integrin alphaV/beta3 at the membrane surface in angiogenic blood vessels and melanomas. Found in mitochondria, along microfibrils, and in nuclei of cardiomyocytes (By similarity).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
Function: Ubiquitinous metalloproteinase that is involved in diverse functions such as remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. As well as degrading extracellular matrix proteins, can also act on several nonmatrix proteins such as big endothelial 1 and beta-type CGRP promoting vasoconstriction. Also cleaves KISS at a Gly-|-Leu bond. Appears to have a role in myocardial cell death pathways. Contributes to myocardial oxidative stress by regulating the activity of GSK3beta. Cleaves GSK3beta in vitro. Involved in the formation of the fibrovascular tissues Catalytic activity: Cleavage of gelatin type I and collagen types IV, V, VII, X. Cleaves the collagen-like sequence Pro-Gln-Gly-|-Ile-Ala-Gly-Gln.Cofactor: Binds 4 calcium ions per subunit
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
