FGF4 ELISA 试剂盒

• 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

• 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

• 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
• Cell lysate buffer

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.

1. Bring all reagents and samples to room temperature (18 - 25°C) before use. 2. Sample dilution: Tissue lysate and cell lysate sample should be diluted at least 5-fold with 1x Sample Diluent Buffer. 3. Sample Diluent Buffer/Assay Diluent should be diluted 5-fold with deionized or distilled water. 4. paration of standard: Briefly spin the vial of Item C. Add 400 µl 1x Sample Diluent Buffer(Item D) into item C vial to prepare a 50 ng/ml standard. Dissolve the powder thoroughly by a gentle mix. Add 180 µl FGF-4 standard from the vial of Item C, into a tube with 320 µl 1x Sample Diluent Buffer to prepare a 18,000 pg/ml stock standard Solution. Pipette 400 µl 1x Sample Diluent Buffer into each tube. Use the stock standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. 1x Assay Diluent 1x Sample Diluent Buffer serves as the zero standard (0 pg/ml). 5. 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 Diuent 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 65-fold with 1x Assay Diuent and used in step 4 of Part VI Assay Procedure. 7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) before use. HRP-Streptavidin concentrate should be diluted 6,000-fold with 1x Assay Diuent. 8. Cell lysate buffer is diluted to 2-fold with deionized or distilled water (for cell lysate and tissue lysate).

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 with plate holder and incubate for 2.5 hours at room temperature or over night at 4°C. 3. Discard the solution and wash 4 times with 1x Wash Solution (200 µl each). 4. Add 100 µl of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature. 5. Discard the solution and wash 4 times with 1x Wash Solution (200 µl each). 6. Add 100 µl of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature. 7. Discard the solution and wash 5 times with 1x Wash Solution (200 µl each). 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. 9. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately.

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.