Intended Use
The CD32 (Phospho-Tyr292) Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor CD32 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CD32 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CD32 phosphorylation.
Contents of Kit
1. 96-Well Cell Culture Clear-Bottom Microplate, 2 Plates
2. 10× TBS, 30 ml (10×), 1 month at 4°C
3. Quenching Buffer, 24 ml (1×), 1 month at 4°C
4. Blocking Buffer, 50 ml (1×), 1 month at 4°C
5. 15× Wash Buffer, 60 ml (15×), 1 month at 4°C
6. 100× Anti-CD32 (Phospho-Tyr292) Antibody, 60 μl (100×), 1 month at 4°C
7. 100× Anti-CD32 Antibody, 60 μl (100×), 1 month at 4°C
8. 100× Anti-GAPDH Antibody, 60 μl (100×), 1 month at 4°C
9. HRP-Conjugated Anti-Rabbit IgG Antibody, 12 ml (1×), 1 month at 4°C
10. HRP-Conjugated Anti-Mouse IgG Antibody, 12 ml (1×), 1 month at 4°C
11. Primary Antibody Diluent, 12 ml (1×), 1 month at 4°C
12. Ready to Use Substrate, 12 ml (1×), 1 month at 4°C
13. Stop Solution, 12 ml (1×), 1 month at 4°C
14. Crystal Violet Solution, 12 ml (1×), 1 month at 4°C
15. SDS Solution, 24 ml (1×), 1 month at 4°C
16. Adhesive Plate Seals, 2 Seals
Standard Curve
Background subtraction
Average the duplicate or triplicate absorbance readings for each control and sample, subtracting them from the averaged absorbance for wells where primary antibodies have been omitted.
Anti-GAPDH Internal positive control
The OD450 values obtained ensure the assay is functioning, and cell densities between wells should be proportionate to seeding concentration. Stimulation of cells should impact target primary antibody absorbance values, with no effect on the anti-GAPDH absorbance.
Crystal Violet Staining Normalization
The Crystal Violet staining method enables intensity normalization within the same well. By using the ratio, the OD450 readings obtained can be normalized with the OD595 values. This constitutes a within-well method of analysis.
If the absorbance was too high and a tenfold dilution was required. Multiply all values by 10 prior to normalization.
a. OD450 anti-CD32 (Phospho-Tyr292)/OD595 Crystal Violet.
b. OD450 anti-CD32/OD595 Crystal Violet.
By doing so, the expression levels of CD32 (Phospho-Tyr292) and CD32 are adjusted to account for the cell density present in each well.
Phosphorylation to Non-Phosphorylation Comparison
After normalization Crystal Violet staining, the resultant proportional values can be used to analyze the effects of stimulants in this assay.
A non-phosphorylated primary antibody is included to normalize the absorbance values between phosphorylated to non-phosphorylated values.
Both antibodies utilize the proportion for analysis as follows:
OD450 (CV Normalized) (CD32 (Phospho-Tyr292))/
OD450 (CV Normalized) (CD32)
Citations
COA
Certificate of Analysis
