General protocol for phospho ELISA kits is as follows. Capture antibodies are precoated on the bottom of a 96-well plate. The cell extract or standard is added to the wells and incubated to allow target proteins to bind. The wells are then washed to remove unbound material, and the detection antibody is added and incubated to form a sandwich around the protein of interest. HRP anti-rabbit antibody is then added and incubated to allow binding to the rabbit-derived detection antibody. Next, the chromogen substrate for HRP is added, and the subsequent enzymatic reaction turns the solution blue. Finally, the reaction is stopped, turning the solution yellow in proportion to the amount of target protein in the sample. Absorbance is read in a microplate reader at 450 nm.
Phospho-Specific ELISA Kits
Measure signaling of intracellular proteins
Phosphorylation-specific Invitrogen™ ELISA kits are high-quality ELISA kits, designed for researchers studying intracellular proteins involved in signaling pathways. These assay kits are designed to deliver accurate, sensitive, and fast protein quantitation of total and phosphorylated, modified, or cleavage site-specific proteins in a broad range of sample types.
In addition to our existing Invitrogen™ phospho ELISA kits, we now also offer Invitrogen™ InstantOne™ ELISA kits. InstantOne kits use a streamlined, easy-to-use, 1-hour, 1-wash procedure to accurately and reliably detect total and phosphorylated targets.
Select phospho-specific ELISA kits from among all target-specific ELISA kits using the following selection guide.
Abnormal phosphorylation states are implicated in many human diseases, including cancer. Studying the phosphorylation and post-translational modification of key protein targets is essential to better understanding the mechanisms behind disease.
We have phosphorylation-specific ELISA kits for many of the key targets and signaling pathways implicated in various diseases, including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2, Caspase 3 signaling. These kits are manufactured to help ensure excellent quality and reproducibility. The kits must meet quality-controlled specifications for sensitivity, dynamic range, precision, specificity, recovery, and lot-to-lot consistency.
Two styles of Invitrogen sandwich assay kits are available:
- Phospho-ELISA kits
- InstantOne ELISA kits
Invitrogen phospho-ELISA kits
These “phosphoELISA” kits use a standard sandwich ELISA format involving pre-coated capture antibodies and stepwise addition thereafter of samples, detector antibodies, and other reagents.
- Specificity—two antibodies directed against the analyte provide better specificity than western blotting
- Sensitivity—more sensitive than western blotting
- Quantitation—get quantitative data in contrast to western blots
- Medium throughput—96-well format, results in 4 hours, no densitometry analysis needed
Choose phospho-specific ELISA kits from among all target-specific ELISA kits using the following selection guide:
InstantOne ELISA kits are new to the Invitrogen portfolio, having previously been sold under the eBioscience brand, now part of Thermo Fisher Scientific. In the InstantOne assay protocol, the target analyte binds to the two sandwich ELISA antibodies in solution while the capture antibody binds to the plate through a proprietary mechanism, so both the sample and the assay reagents may be added to the InstantOne ELISA assay plate at the same time. Unbound assay reagents and nonspecific sample components are washed away (just as in a traditional sandwich ELISA), while the specific analyte is detected through a colorimetric detection.
- Fast—less than 1/3 the time of traditional ELISAs; 1 hour and 1 wash protocol
- Flexible—enables the detection of total and/or phospho protein levels of a single protein up or down a pathway on a single plate
- Sensitive—meets or exceeds industry standards with only 2-3,000 cells needed per well
InstantOne ELISA assays use the traditional sandwich ELISA format, but with a major difference. InstantOne allows for greater flexibility, ease of use, and reduced assay time by allowing the target analyte to bind to both of the two sandwich ELISA antibodies in solution as the capture antibody binds to the plate through a proprietary mechanism. This allows for both the sample and the assay reagents to be added to the InstantOne ELISA assay plate at the same time. Unbound assay reagents and non-specific sample components are washed away just as in a traditional sandwich ELISA, while the specific analyte is detected though a colorimetric detection. The entire process takes just over an hour to complete.
In addition to the ease that the 1-hour/1-wash InstantOne ELISA provides, it also adds a layer of flexibility not readily accessible with traditional sandwich ELISAs. As the antibodies are not pre-coated in the wells, several different targets can be analyzed simultaneously on the same plate in different wells. Simply add the sample lysate to the plate wells and add different antibody reagent cocktails to the different wells. Analysis of both total and phosphorylated targets across family members or down pathways in the same plate has never been easier.
Summary of the Invitrogen InstantOne ELISA kit procedure.
Choose InstantOne ELISA kits from among all target-specific ELISA kits using the following selection guide:
For Research Use Only. Not for use in diagnostic procedures.