ERK3 (Phospho-Ser189) ELISA Kit

Sample

Cultured cells

Species Reactivity

Human; Mouse; Rat

Detection Method

sELISA

Intended Use

The Phospho-ERK3(Ser189) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Phospho-ERK3(Ser189) protein expression profile in cells. The kit can be used for measuring the relative amounts of Phospho-ERK3(Ser189) in cultured cells as well as screening for the effects that variuos treatments, inhibitors (ie siRNA or chemicals), or activators have on Phospho-ERK3(Ser189).

Contents of Kit

1. 96-Well Cell Culture Clear-Bottom Microplate, 1 Plate
2. 10× TBS, 30 ml (10×)
3. Quenching Buffer, 24 ml (1×)
4. Blocking Buffer, 50 ml (1×)
5. 15× Wash Buffer, 60 ml (15×)
6. 100× Anti-ERK3 (Phospho-Ser189) Antibody , 60 μl (100×)
7. 100× Anti-GAPDH Antibody, 60 μl (100×)
8. HRP-Conjugated Anti-Rabbit IgG Antibody, 6 ml (1×)
9. HRP-Conjugated Anti-Mouse IgG Antibody, 6 ml (1×)
10. Primary Antibody Diluent, 12 ml (1×)
11. Ready to Use Substrate, 12 ml (1×)
12. Stop Solution, 12 ml (1×)
13. Crystal Violet Solution, 6 ml (1×)
14. SDS Solution, 24 ml (1×)
15. Adhesive Plate Seals, 2 Seals

Storage

2-8°C

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-ERK3 (Phospho-Ser189)/OD595 Crystal Violet.
b. OD450 anti-ERK3/OD595 Crystal Violet.
By doing so, the expression levels of ERK3 (Phospho-Ser189) and ERK3 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) (ERK3 (Phospho-Ser189)) / OD450 (CV Normalized) (ERK3)