Components
96-Well Cell Culture Clear-Bottom Microplate: 2 Plates
TBS: 24 mL (10x), Clear
Quenching Buffer: 24 mL (1x), Clear
Blocking Buffer: 50 mL (1x), Clear
Wash Buffer: 50 mL (10x), Clear
Anti-CXCR4 (Phospho-Ser339) antibody (Rabbit Polyclonal): 60 µl (100x), Red
Anti-CXCR4 antibody (Rabbit Polyclonal): 60 µl (100x), Purple
Anti-GAPDH antibody (Mouse Monoclonal): 60 µl (100x), Green
HRP-Conjugated Anti-Rabbit IgG antibody 12 mL (1x), Glass
HRP-Conjugated Anti-Mouse IgG antibody 12 mL (1x), Glass
Primary antibody Diluent: 12 mL (1x), Clear
Ready-to-Use Substrate: 12 mL (1x), Brown
Stop Solution: 12 mL (1x), Clear
Crystal Violet Solution: 12 mL (1x), Glass
SDS Solution: 24 mL (1x), Clear
Adhesive Plate Seals: 4 Seals
Material not included
The following materials and equipment are NOT provided in this kit but are necessary to successfully conduct the experiment: Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional). Micropipettes with capability of measuring volumes ranging from 1 µL to 1 mL 37% formaldehyde.
Reagent Preparation
Fixing Solution: It is prepared by adding formaldehyde to 1x TBS with light mixing. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
Quenching Buffer: Quenching Buffer is used to inactivate the endogenous peroxidase activity of the seeded cells.
Blocking Buffer: Blocking Buffer is used to block additional binding sites in each well.
Wash Buffer: 1x Wash Buffer can be prepared by adding 1 volume of 10x Wash Buffer provided in the kit to 9 volumes of ddH2O.
100x Anti-CXCR4 (Phospho-Ser339) antibody: This antibody is a rabbit polyclonal antibody. This antibody was tested to be specific for the CXCR4 protein phosphorylated at Ser339.
100x Anti-CXCR4 antibody: This antibody is a rabbit polyclonal antibody. This antibody was tested to be specific for the CXCR4 protein.
Assay Procedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker.
7.Add 100 µL Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
11.Add 50 µL of 1x primary antibodies (Anti-CXCR4 (Phospho- Ser339) antibody, Anti-CXCR4 antibody and/or Anti-GAPDH antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
13.Add 50 µL of 1x secondary antibodies to corresponding wells and incubate for 1.5 hours at room temperature with gentle shaking on the shaker.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark with gentle shaking on the shaker.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.
17.After finishing reading the absorbance at 450 nm, wash the plate twice with 200 µL of Wash Buffer and twice with 200 µL of 1x TBS for 5 minutes each. Tap the plates on paper towel to remove the excess liquid. Let plate air dry for 5 minutes at room temperature.
18.Add 50 µL of Crystal Violet Solution to each well, incubate for 30 minutes at room temperature on the shaker.
19.Flick the plate to remove Crystal Violet Solution, rinse the plate by filling the wells with running tap water, and wash the plate with 200 µL of 1x TBS 3 times, 5 minutes each with gently shaking on the shaker.
20.Add 100 µL of SDS Solution into each well and incubate on the shaker at room temperature for 1 hour.
21.Read absorbance at 595 nm with microplate reader.
Calculation of Results
Anti-CXCR4 antibody normalization: The OD values obtained for the phosphorylated target protein can be normalized using the OD values obtained for the non-phosphorylated target protein via the proportion, OD450 (Anti-CXCR4 P-Ser339 antibody)/OD450 (Anti-CXCR4 antibody).
GAPDH normalization: The OD450 values obtained for the target protein (phosphorylated and non- phosphorylated) can be normalized using the OD450 values obtained for GAPDH. Crystal Violet Staining normalization: The measured OD450 readings can be normalized using the OD595 values via the proportion, OD450/OD595.
Assay Precision
This ELISA kit is intended for research purposes only, NOT for diagnostic or clinical procedures of any kind.
Precaution of Use
Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Personal protection is strongly recommended while working with this chemical.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material.
Crystal Violet is an intense stain reagent. Avoid contact with clothing.
Storage Comment
Upon receipt, the kit should be stored at 4°C. The un-opened kit will be stable for up to 6 months from the date of shipment if stored at 4°C. Diluted Anti-CXCR4 (Phospho-Ser339) antibody, Anti-CXCR4 antibody and diluted Anti-GAPDH antibody can each be stored at 4°C for up to two weeks. HRP-Conjugated Anti-Rabbit IgG antibody and HRP-Conjugated Anti- Mouse IgG antibody will be stable at 4°C for up to six months. The SDS Solution should be stored at room temperature or warmed up to room temperature if stored at 4°C.
Note
Upon receipt, the kit should be stored at 4°C. The un-opened kit will be stable for up to 6 months from the date of shipment if stored at 4°C. Diluted Anti-CXCR4 (Phospho-Ser339) antibody, Anti-CXCR4 antibody and diluted Anti-GAPDH antibody can each be stored at 4°C for up to two weeks. HRP-Conjugated Anti-Rabbit IgG antibody and HRP-Conjugated Anti- Mouse IgG antibody will be stable at 4°C for up to six months. The SDS Solution should be stored at room temperature or warmed up to room temperature if stored at 4°C.
Restrictions
For Research Use only
Datasheet