Sonic Hedgehog ELISA Kit

Catalog No. ABIN625086

Product Details Sonic Hedgehog ELISA Kit

Target: Sonic Hedgehog (SHH)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 8-10000 pg/mL

Minimum Detection Limit: 8 pg/mL

Application: ELISA

Purpose: Human Sonic Hedgehog N-Terminal (Shh-N) 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 the following cytokines tested: human Angiogenin, BDNF, BLC, ENA-78, FGF- 4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin (OB), MCP-1, MCP-2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

Sensitivity: 42 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: Assay Diluent C (Item L) is used for dilution of serum/plasma samples, 1x Assay Diluent B (Item E) can be used for dilution of cell culture supernates/urine. 3. Assay Diluent B should be diluted 5-fold with deionized or distilled water before use. 4. Preparation of standard: Briefly spin the vial of Item C and then add 320 µl Assay Diluent C (for serum/plasma samples) or 1x Assay Diluent B (for cell culture supernates/urine) into Item C vial to prepare a 50 ng/ml standard. Dissolve the powder thoroughly by a gentle mix. Add 100 µl Shh-N standard (50 ng/ml) from the vial of Item C, into a tube with 400 µl Assay Diluent C to prepare a 10,000 pg/ml standard solution. Pipette 300 µl Assay Diluent C into each tube. Use the 10,000 pg/ml standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. Gently vortex to mix. Assay Diluent C serves as the zero standard (0 pg/ml). The 10,000 pg/ml standard in Assay Diluent B (for cell culture supernates/urine) will be saturated, we recommended starting from 2,000 pg/ml as the highest concentration and producing a series dilution (800, 320, 128, 51.2, 20.48, 8.19, 0 pg/ml) using 1x Assay Diluent B. 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 10,000-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 2 µl of HRP-Streptavidin concentrate into a tube with 198 µl 1x Assay Diluent B to prepare a 100-fold diluted HRP- Streptavidin solution (do not store the diluted solution for next day use). Mix through and then pipette 100 µl of prepared 100-fold diluted solution into a tube with 10 ml 1x Assay Diluent B to prepare a final 10,000 fold diluted HRP-Streptavidin solution.

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 µl) 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 3. 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 3. 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.

Restrictions: For Research Use only

Handling

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 Sonic Hedgehog ELISA Kit (ABIN625086)

Cigna, Farrokhi Moshai, Brayer, Marchal-Somme, Wémeau-Stervinou, Fabre, Mal, Lesèche, Dehoux, Soler, Crestani, Mailleux: "The hedgehog system machinery controls transforming growth factor-?-dependent myofibroblastic differentiation in humans: involvement in idiopathic pulmonary fibrosis." in: The American journal of pathology, Vol. 181, Issue 6, pp. 2126-37, (2012) (PubMed).

Target Details for Sonic Hedgehog

Target: Sonic Hedgehog (SHH)

Alternative Name: Sonic Hedgehog (SHH Products)

Synonyms: HHG1 ELISA Kit, HLP3 ELISA Kit, HPE3 ELISA Kit, MCOPCB5 ELISA Kit, SMMCI ELISA Kit, TPT ELISA Kit, TPTPS ELISA Kit, 9530036O11Rik ELISA Kit, Dsh ELISA Kit, Hhg1 ELISA Kit, Hx ELISA Kit, Hxl3 ELISA Kit, M100081 ELISA Kit, fc83d08 ELISA Kit, shh ELISA Kit, syu ELISA Kit, vhh-1 ELISA Kit, vhh1 ELISA Kit, wu:fc83d08 ELISA Kit, Xhh ELISA Kit, hedgehog ELISA Kit, xshh ELISA Kit, SHH ELISA Kit, twh ELISA Kit, twhh ELISA Kit, sonic hedgehog ELISA Kit, sonic hedgehog a ELISA Kit, sonic hedgehog L homeolog ELISA Kit, sonic hedgehog protein A ELISA Kit, sonic hedgehog b ELISA Kit, SHH ELISA Kit, Shh ELISA Kit, shha ELISA Kit, shh.L ELISA Kit, shh ELISA Kit, shhb ELISA Kit

Background: The Human Shh-N (Sonic Hedgehog N Terminus) ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human Shh-N in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human Shh-N coated on a 96-well plate. Standards and samples are pipetted into the wells and Shh-N present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human Shh-N antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Shh-N bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Gene ID: 6469

UniProt: Q15465

Pathways: Hedgehog Signaling, Dopaminergic Neurogenesis, Regulation of Muscle Cell Differentiation, Tube Formation, Skeletal Muscle Fiber Development