TIMP1 ELISA Kit

Catalog No. ABIN625214

Product Details TIMP1 ELISA Kit

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

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 4-3000 pg/mL

Minimum Detection Limit: 4 pg/mL

Application: ELISA

Purpose: Rat TIMP-1 ELISA Kit for cell culture supernatants, plasma, and serum samples.

Sample Type: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificity: This ELISA kit shows no cross-reactivity with any of the cytokines tested: rat CINC-2, CINC-3, CNTF, Fractalkine, IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, LIX, Leptin, MCP-1, MIP-3 alpha, beta- NGF, TNF-alpha, VEGF.

Cross-Reactivity (Details): This ELISA kit shows no cross-reactivity with any of the cytokines tested (e.g., rat CINC-2, CINC-3, CNTF, Fractalkine, IL-1alpha, IL- 1beta, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, LIX, Leptin, MCP-1, MIP-3alpha, beta- NGF, TNF-alpha, VEGF)

Sensitivity: < 4 pg/mL

Characteristics:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Material not included:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Application Details

Application Notes: Recommended Dilution for serum and plasma samples30 - 300 fold

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Reagent Preparation:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of culture supernatants. Suggested dilution for normal serum/plasma: 30-300 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium, Assay Diluent B should be diluted 5-fold with deionized or distilled water before use ) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 30 µL TIMP-1 standard from the vial of Item C, into a tube with 470 µL Assay Diluent A or 1x Assay Diluent B to prepare a 3,000 pg/mL stock standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 30 µL standard + 470 µL 3,000 1,000 333.3 111.1 37.0 12.3 4.1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 400-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 30 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a 400-fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Assay Procedure:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette 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.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Rat TIMP-1 concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10 Assay Diluent B Rat TIMP-1 concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of TIMP-1 is typically less than 4 pg/mL.
Recovery: Recovery was determined by spiking various levels of rat TIMP-1 into rat serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 92.79 85-104 Plasma 94.65 84-103 Cell culture media 93.26 86-104
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 93 93 94 Range ( %) 83-102 84-104 84-103 1:4 Average % of Expected 90 92 101 Range ( %) 84-104 85-102 88-107
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Assay Precision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Expiry Date: 6 months

References for TIMP1 ELISA Kit (ABIN625214)

Tseng, Tsai, Yu, Fu, Huang, Cheng: "Comparative effects of bone marrow mesenchymal stem cells on lipopolysaccharide-induced microglial activation." in: Oxidative medicine and cellular longevity, Vol. 2013, pp. 234179, (2013) (PubMed).

Shen, Wang, Chou, Liou, Yen, Wang, Liao: "Dimemorfan protects rats against ischemic stroke through activation of sigma-1 receptor-mediated mechanisms by decreasing glutamate accumulation." in: Journal of neurochemistry, Vol. 104, Issue 2, pp. 558-72, (2008) (PubMed).

Rohrbach, Martin, Niemann, Cherubini: "Enhanced myocardial vitamin C accumulation in left ventricular hypertrophy in rats is not attenuated with transition to heart failure." in: European journal of heart failure, Vol. 10, Issue 3, pp. 226-32, (2008) (PubMed).

Omi, Mochida, Iwashina, Katsuno, Hiyama, Watanabe, Serigano, Iwabuchi, Sakai: "Low-intensity pulsed ultrasound stimulation enhances TIMP-1 in nucleus pulposus cells and MCP-1 in macrophages in the rat." in: Journal of orthopaedic research : official publication of the Orthopaedic Research Society, Vol. 26, Issue 6, pp. 865-71, (2008) (PubMed).

Lijnen, Papparella, Petrov, Semplicini, Fagard: "Angiotensin II-stimulated collagen production in cardiac fibroblasts is mediated by reactive oxygen species." in: Journal of hypertension, Vol. 24, Issue 4, pp. 757-66, (2006) (PubMed).

Gomez, Alonso, Yoshiji, Thorgeirsson: "Tissue inhibitors of metalloproteinases: structure, regulation and biological functions." in: European journal of cell biology, Vol. 74, Issue 2, pp. 111-22, (1998) (PubMed).

Hayakawa, Yamashita, Tanzawa, Uchijima, Iwata: "Growth-promoting activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) for a wide range of cells. A possible new growth factor in serum." in: FEBS letters, Vol. 298, Issue 1, pp. 29-32, (1992) (PubMed).

Target Details for TIMP1

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

Alternative Name: TIMP-1 (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: Metalloproteinase inhibitor 1 (Tissue inhibitor of metalloproteinases 1) (TIMP-1)

Gene ID: 116510

UniProt: P30120