TIMP1 ELISA Kit

Catalog No. ABIN579240

Product Details TIMP1 ELISA Kit

Target: TIMP1 (TIMP Metallopeptidase Inhibitor 1 (TIMP1))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.156-10 ng/mL

Minimum Detection Limit: 0.156 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of Rat tissue inhibitors of metalloproteinase,TIMP-1 concentrations in cell culture supernates, serum, and plasma.

Sample Type: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural Rat TIMP-1.

Characteristics: Rattus norvegicus,Rat,Metalloproteinase inhibitor 1,Tissue inhibitor of metalloproteinases 1,TIMP-1,Timp1,Timp-1

Components: Reagent (Quantity ): Assay plate (1), Standard 2 Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent B (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1 x 10ml) Stop Solution (1 x 10ml)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TIMP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TIMP-1 present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for TIMP-1 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color 2 develops in proportion to the amount of TIMP-1 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 10 ng/mL. Allow the standard to sit for about 10 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the highest standard (10 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). ng/mL 10 5 2.5 1.25 0.625 0.312 0.156 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A or B (1:100), respectively.

Sample Collection: Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000 g. Remove serum and assay immediately or aliquot and store samples at -20 or -80 . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 g at 2 - 8 within 30 minutes of collection. Store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. 3 Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Note: Serum and plasma to be used within 7 days may be stored at 2-8, otherwise samples must stored at -20 ( 1 month) or -80 ( 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

Assay Procedure:

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 4 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 4 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 particular experiments.
1. Add 100 of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for two hours at 37 .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μ l) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 μ l of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for one hour at 37 .
6. Repeat the aspiration/wash process for five times as conducted in step
4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 30 minutes at 37 . Protect from light.
8. Add 50 μ l of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between 5 incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the TIMP-1 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for TIMP1

Target: TIMP1 (TIMP Metallopeptidase Inhibitor 1 (TIMP1))

Alternative Name: Timp1 (TIMP1 Products)

Synonyms: TIMP-1 ELISA Kit, TIMP1 ELISA Kit, DKFZp468A0912 ELISA Kit, CLGI ELISA Kit, EPA ELISA Kit, EPO ELISA Kit, HCI ELISA Kit, TIMP ELISA Kit, Timp ELISA Kit, Clgi ELISA Kit, TIMP metallopeptidase inhibitor 1 ELISA Kit, tissue inhibitor of metalloproteinase 1 ELISA Kit, TIMP1 ELISA Kit, Timp1 ELISA Kit

Background: Matrix metalloproteinases (MMPs), also called matrixins, constitute a family of zinc and calcium dependent endopeptidases that function in the breakdown of extracellular matrix and in the processing of a variety of biological molecules. They play an important role in many normal physiological processes such as embryonic development, morphogenesis, reproduction and tissue remodeling.They also participate in many pathological processes such as arthritis, cancer and cardiovascular disease.While the amounts of newly synthesized MMPs are regulated mainly at the levels of transcription, the proteolytic activities of existing MMPs are controlled through both the activation of proenzymes or zymogens and the inhibition of active enzymes by endogenous inhibitors such as-macroglobulins and tissue inhibitors of alpha2 metalloproteinases (TIMPs). The mammalian TIMP family includes four members that share structural similarity.All TIMP proteins have 12 conserved cysteine residues that form six intrachain disulfide bonds, resulting in an extremely stable protein with six loops. The TIMP protein has two structurally and functionally distinct domains: the N-terminal domain consisting of loops 1 - 3 that are responsible for tight, but non-covalent binding to the active MMPs in a 1:1 stoichiometry, and the C-terminal domain consisting of loops 4 - 6 that enhances the enzyme-inhibitor interactions. In the case of TIMP-1, the C-terminal domain has also been shown to bind the hemopexin-like domain of pro MMP-9. TIMP-1 stimulates erythropoiesis, inhibits angiogenesis and is an anti-apoptotic agent for B cells. These TIMP-1 functions may be independent of MMP inhibition . Rat TIMP-1 is a 28 - 35 kDa secreted glycoprotein . The protein is synthesized as a 217 amino acid (aa) precursor that contains a 23 aa signal peptide and a 194 aa mature form. TIMP-1 is widely expressed in many cells such as fibroblasts, osteoblasts, endothelial cells, granulosa cells, dendritic cells, vascular smooth muscle cells, adipocytes, and monocytes.

Gene ID: 3132