Anti-EDG7 antibody (ab23692)

Application notes

IHC-Fr: Use at an assay dependent dilution (PMID 18772233).
WB: Use at a concentration of 2.5 µg/ml. Detects a band of approximately 40 kDa (Predicted molecular weight: 40 kDa). Block with 0.5% non-fat dry milk in TBS pH 7.4) for 1-2 hours at room temperature with gentle shaking or overnight at 4 degrees C. Primary antibody dilutions should be carried out in 0.5% non-fat dry milk in TBS pH 7.4 plus 0.1% Tween-20 or a similar buffer. Membranes should be incubated in the primary antibody for at least 1 hour. Increased sensitivity may be achieved by incubating for longer periods of time (at room temp. or 16-18 hrs at 4 degrees C.)

ICC/IF: Use at a concentration of 2.5 µg/ml.
Protocol:
1. Grow cells in 6 or 24 well plates until confluent.
2. Wash briefly with TBS, pH 7.4.
3. Fix the cells with 1% formaldehyde in TBS, pH 7.4, for 10 minutes.
4. Wash the cells 3 times with TBS containing 0.1% Triton-X 100 (TBSTX), 10 minutes each.
5. Incubate the cells with 10% normal serum (from the same species in which the secondary antibody is raised) in TBSTX for 30 minutes.
6. Incubate the cells with the primary antibody for 1 hour (recommended starting concentration of 2.5 µg/ml. The optimal working condition should be determined by titration).
7. Wash the cells 3 times with TBSTX, 10 minutes each.
8. Incubate the cells in the dark for 1 hour with a fluorochrome-conjugated secondary antibody at a concentration recommended by the provider.
9. Wash the cells 3 times with TBSTX, 10 minutes each.
Examine the staining under a fluorescent microscope with an appropriate filter. Store the plate at 4°C in dark for later analysis.


Not tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.