RAGE ELISA Kit

Catalog No. ABIN1980069

Product Details RAGE ELISA Kit

Target: RAGE (AGER) (Advanced Glycosylation End Product-Specific Receptor (AGER))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 300 pg/mL-300 ng/mL

Minimum Detection Limit: 300 pg/mL

Application: ELISA

Purpose: Rat RAGE ELISA Kit for cell culture supernatants, plasma, and serum samples.

Sample Type: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificity: Cross Reactivity: This ELISA kit shows no cross-reactivity with the following 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, Leptin, Lix, MCP-1, MIP-3 alpha, beta-NGF, TIMP-1, TNF-alpha.

Sensitivity: 300 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 samples2 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 D (Item K) should be used for dilution of serum/plasma/culture supernatants. Suggested dilution for normal serum/plasma: 3-30 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 D (Item K) and Assay Diluent B (Item E) should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 500 µL 1x Assay Diluent D (Item K) into Item C vial to prepare a 300 ng/mL standard solution. Dissolve the powder thoroughly by a gentle mix. Pipette 400myl 1x Assay Diluent D into each tube. Use the 300 ng/mL standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Assay Diluent D serves as the zero standard (0 ng/mL). 200 µL 200 µL 200 µL 200 µL 200 µL 200myl Item C vial + 500 µL 300 100 33.33 11.11 3.70 1.23 0.41 0 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/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 (Item E) 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 Band 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 (Item E). 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. Rat RAGE concentration (ng/mL) 0.1 1 10 100 1000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent D
Sensitivity: The minimum detectable dose of RAGE is typically less than 300 pg/mL.
Recovery: Recovery was determined by spiking RAGE into normal rat serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 79.40 70-91 Plasma 74.84 69-88 Cell culture media 64.74 53-70
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 136.3 145.6 133.6 Range ( %) 122-144 133-152 120-143 1:4 Average % of Expected 137.2 143.5 134.1 Range ( %) 123-144 132-150 120-144
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 RAGE ELISA Kit (ABIN1980069)

Ibrahim, El-Denshary, Abdallah: "Geraniol, alone and in combination with pioglitazone, ameliorates fructose-induced metabolic syndrome in rats via the modulation of both inflammatory and oxidative stress status." in: PLoS ONE, Vol. 10, Issue 2, pp. e0117516, (2015) (PubMed).

Abdelsalam, Safar: "Neuroprotective effects of vildagliptin in rat rotenone Parkinson's disease model: role of RAGE-NF?B and Nrf2-antioxidant signaling pathways." in: Journal of neurochemistry, Vol. 133, Issue 5, pp. 700-7, (2015) (PubMed).

Greco, Tassorelli, Mangione, Levandis, Certo, Nappi, Bagetta, Blandini, Amantea: "Neuroprotection by the PARP inhibitor PJ34 modulates cerebral and circulating RAGE levels in rats exposed to focal brain ischemia." in: European journal of pharmacology, Vol. 744, pp. 91-7, (2014) (PubMed).

Park, Kim, Jung, Chung, Hasham, Park, Choi: "A long-standing hyperglycaemic condition impairs skin barrier by accelerating skin ageing process." in: Experimental dermatology, Vol. 20, Issue 12, pp. 969-74, (2011) (PubMed).

Target Details for RAGE

Target: RAGE (AGER) (Advanced Glycosylation End Product-Specific Receptor (AGER))

Alternative Name: RAGE / AGER (AGER Products)

Synonyms: RAGE ELISA Kit, AGER ELISA Kit, advanced glycosylation end-product specific receptor ELISA Kit, advanced glycosylation end product-specific receptor ELISA Kit, MAPK/MAK/MRK overlapping kinase ELISA Kit, AGER ELISA Kit, Ager ELISA Kit, LOC719012 ELISA Kit

Background: Advanced glycosylation end product-specific receptor (Receptor for advanced glycosylation end products)

UniProt: Q63495

Pathways: Carbohydrate Homeostasis, Toll-Like Receptors Cascades, Smooth Muscle Cell Migration, S100 Proteins