His Tag ELISA Kit (Advanced)

Sample Preparation
Cell culture supernatants or lysed cell homogenate supernatants were centrifuged at 2000 × g for 10 minutes to remove debris.
The supernatant can be loaded directly after collection or diluted with Assay Diluent.
Store undiluted samples at or below-20 °C.
Avoid repeated freeze-thaw cycles.

Enzyme plate preparation
The 96-well detachable enzyme plate is ready to use and can be loaded directly without other processing.
The plate type is 12 1 x 8 slats.
Unused slats should be immediately returned to the foil pouch containing the desiccant package, resealed and stored at 2-8 °C.

Experimental procedure
All materials and reagents will need to be equilibrated to room temperature before use.
It is recommended that all standards, controls and samples be tested in duplicate in parallel wells.
1) Prepare all reagents, working standards and samples according to the instructions on the previous page.
2) Remove the excess enzyme labeled strips from the removable plate frame, place them back in the foil bag containing the desiccant package, reseal and return to storage at 2-8 º C.
3) Take 50 µ L of sample or standard and add to the corresponding well.
4) In each well 50 µ L of 1X HRP conjugated anti-His tag Antibody.
5) Apply a sealing film and incubate for 45 minutes on an enzyme label plate constant temperature shaker with the setting conditions of "rotational speed of 500 rpm and temperature of 25 °C".
6) Wash each well with 300 µ L of 1X Wash Buffer three times.
After the last wash, turn the board upside down and gently pat it several times on a clean paper towel to remove any excess liquid.
7) Add 100 µ L of TMB substrate to each well, and develop color for 10 minutes on a 500 rpm plate thermostatic shaker protected from light.
Given the variability of laboratory environmental conditions, the optimal color development time may vary between 5 and 15 minutes.
Note: After adding the stop solution, the color changes from blue to yellow, and the signal intensity is increased by about 3 times.
To avoid signal saturation, you can pre-read the plate before adding the stop solution, and proceed to the next operation before the high concentration of the standard reaches blue O.D.650 = 1.0.
8) Add 100 µ L of stop solution to each well, mix well and determine the OD450 of each well within 30 minutes using a microplate reader set at 450 nm.
If wavelength correction is available, set to 540nm or 570nm and the calibrated OD value is the measured value of 450 nm minus the measured value of 540 nm or 570 nm.

Results Calculation
Take the average value of replicate readings of each standard and sample and record it as the B value of the standard or sample, and record the average value of replicate readings of zero in the standard curve column as the B0 value of this experiment.
Convert the OD value to Percent Bound (B/B0), i.e. (B/B0) × 100%
Standard curves and scaled sample concentrations were created by using computer software capable of generating four-parameter logic (4-PL) curve fits.
If the sample is diluted and then loaded, the concentration converted from the standard curve must be multiplied by the dilution factor to obtain the actual concentration of the sample.