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Measure signaling of intracellular proteins

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 ELISA kits specific for the phosphorylation states for many of the key targets and signaling pathways implicated in various diseases, including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2, and Caspase 3 signaling. These kits are available in two different formats–standard sandwich ELISA and InstantOne ELISA kits. Both type of 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.

Invitrogen precoated phospho-ELISA kits

These phospho-ELISA kits use a standard sandwich ELISA format involving pre-coated capture antibodies and stepwise addition thereafter of samples, detector antibodies, and other reagents. The kits come with standard proteins to generate a standard curve for quantitative results.

Features:

  • Quantitation—get quantitative data to complement western blot images
  • Specificity—two antibodies directed against the analyte provide better specificity than western blotting
  • Sensitivity—more sensitive than western blotting with only typically 2,000–3,000 cells needed
  • Medium throughput—96-well format, results in 4 hours, no densitometry analysis needed

Phospho ELISA kit details

Summary of Invitrogen precoated Sandwich ELISA kit protocol

A 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.

How-to videos for precoated Phospho-ELISA kits

Phospho ERK ELISA kit demo video

Learn how to use the phosphorylated ERK 1/2 ELISA Kit

This video demonstrates how to use the precoated sandwich ELISA for phospho-ERK 1 and 2.

Phospho p38 ELISA kit demo video

Learn how to use the phosphorylated p38 ELISA Kit

This video demonstrates how to use the precoated sandwich ELISA for phospho-p38.

Popular standard phospho-ELISA kits

Invitrogen InstantOne Phospho-ELISA kits

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.

Features:

  • Flexible—enables the detection of total and/or phospho protein levels of a single protein up or down a pathway on a single plate
  • Specificity—two antibodies directed against the analyte provide better specificity than western blotting
  • Sensitivity—more sensitive than western blotting with only typically 2-3000 cells needed
  • High throughput—1 hour and 1 wash protocol

InstantOne ELISA Kit assay principle

Summary of the Invitrogen InstantOne ELISA kit procedure.
Summary of the Invitrogen InstantOne ELISA kit procedure.

InstantOne ELISA assays use the traditional sandwich ELISA format, but with a major difference. InstantOne allows for ease of use and reduced hands-on time and 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.

Popular Phospho InstantOne ELISA kits

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