Introduction

“PhosphoELISA” kits are phospho-specific sandwich ELISAs that allow quantitative measurement of intracellular proteins. These ready-to-use kits can be used in signaling studies to measure phosphorylation, post-translational modification (PTM), cleavage site-specific, and total proteins. Validated for quality and reproducibility, each kit meets stringent specifications for sensitivity, dynamic range, precision, specificity, recovery, and lot-to-lot consistency. Our Invitrogen ELISA kits also provide specific detection of phosphorylation and correlate well with western blot data.

Learn more about Phospho ELISA kits: Cell Signaling ELISA Kits and Multiplex Immunoassays

Note: The ELISA assay protocol provided is representative of most phospho-specific ready-to-use ELISA kits. Protocols for individual kits may differ. Depending on the protein of interest, antibodies, buffers, or substrates being used, this general protocol may need to be optimized.

Search available Phospho-specific ELISA kits  Browse available ELISA buffers

Materials

Typical Phospho-specific ELISA kit components

  • Antibody-coated 96-well microplate
  • Detection antibody
  • Standard
  • Standard Diluent Buffer
  • HRP conjugate (antibody or streptavidin)
  • HRP Diluent
  • Wash buffer
  • Chromogenic substrate (TMB)
  • Stop solution
  • Adhesive Plate covers

Additional materials required

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Protocol

Protocol Run time: 4 hours—30 minutes hands-on time.
Note: A standard curve must be run with each assay for quantification.
  1. Carefully read the product information sheet provided with the kit and create a plate layout (include blanks, standard, controls and sample).
  1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
  1. Add 50-100 µL of prepared standard and sample to the wells. Tap gently on the side of the plate to mix. Cover the plate with Adhesive plate cover and incubate at room temperature for 2 hours.

    Note: Samples prepared in Cell extraction buffer must be diluted 1:10 or greater in Diluent buffer. The dilution chosen should be optimized for each experimental system.
  1. Thoroughly aspirate the solution from the wells and discard the liquid.
  1. Wash the wells 4 times with 1X Wash buffer using a squirt wash bottle or an automated 96-well plate washer.

    Note: No matter your method of washing (squirt bottle or automated plate washer), fill wells with at least 400 µL of diluted wash buffer. Let it soak for 15–30 seconds, then decant the liquid. Tap the plate on clean absorbent paper to remove excess solution. Repeat. Use this method for all subsequent wash steps.
  1. Add 100 µL of diluted Detection antibody to the wells. Tap gently on the side of the plate to mix. Cover the plate with Adhesive plate cover and incubate at room temperature for 1 hour.
  1. Thoroughly aspirate or decant solution from wells and discard the liquid.
  1. Wash the wells 4 times with 1X Wash buffer.
  1. Add 100 µL of diluted HRP conjugate to each well. Cover the plate and incubate at room temperature for 30 minutes.
  1. Thoroughly aspirate or decant the solution from wells and discard the liquid.
  1. Wash the wells 4 times with 1X Wash buffer.
  1. Add 100 µL of Chromogenic substrate to each well. The substrate solution begins to turn blue.
  1. Incubate plate for 30 minutes at room temperature in the dark.
  1. Add 100 µL of Stop solution to each well. Tap side of plate gently to mix. The solution in the wells changes from blue to yellow.
  1. Read the absorbance of each well at 450 nm. The plate must be evaluated within 30 minutes after adding the Stop solution.
  1. Use curve-fitting statistical software to plot a 4-parameter logistic curve fit to the standards and then calculate results for the test samples. See ELISA Data Analysis.

Note: When using an Antibody Pair Kit to build your own ELISA assay, coat the capture antibody onto a 96-well microplate. You will need a 96-well microplate, capture antibody, and blocking buffer (usually BSA or milk diluted in PBS). Please follow these steps before starting the protocol above:

  1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
  1. Add 100 µL of diluted Capture antibody to each well. Cover plate and incubate at 4°C overnight.
  1. Bring the plate to room temperature. Thoroughly aspirate or decant the solution from the wells and discard the liquid.
  1. Wash the wells 4 times with Wash buffer.
  1. Add 200 µL of Blocking buffer to each well. Cover plate and incubate at room temperature for 1 hour.
  1. Thoroughly aspirate the solution from the wells and discard the liquid.

    --Continue with above protocol --
Ordering information for related products
Catalog # Name Size Price (USD) Qty
FNN0011 Cell Extraction Buffer Each
185.65

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193.00
Save 7.35 (4%)

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Stylesheet for Classic Wide Template adjustments

For Research Use Only. Not for use in diagnostic procedures.