Product FAQ

How is the phosphorylation state of a protein assessed in the phosphoELISA assay and normalized with the Total ELISA?

Answer

We are proud to offer an expanding line of ELISA assays to quantify the phosphorylation states of various proteins involved in signal transduction. The total ELISA kit quantifies the mass of protein per sample and the phosphoELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) increase, decrease, or remain constant between various samples.

Our method involves the use of two sandwich ELISA assays. Both assays capture total protein, regardless of phosphorylation state, to the wells of an ELISA plate. This is done by coating the plates with a "pan" antibody that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein, and does not block the phosphorylation site to be studied. One assay then quantifies the total amount of protein present in the sample by using a second "pan" antibody for detection of protein regardless of phosphorylation state. This measurement is given in picograms of protein/mL of sample, or sometimes nanograms of protein/mL. The measurement is given in mass units. This is because standards of known mass are available for use in the standard curve for the total assay.

A second assay quantifies the amount of that same protein that has been phosphorylated at a specific amino acid. Instead of a second "pan" antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated at a particular amino acid position. This procedure is extremely similar to an immunoprecipitation reaction in which two different antibodies are used; one to capture a protein from a solution, and one to detect the subset of that protein population that is phosphorylated at a certain site. The ELISA assay has several advantages over immunoprecipitation, including ease of use and increased sensitivity. ELISA is also quantitative. This second assay will detect and quantify only the phosphorylated form of a particular protein. This second measurement is given in "units", which we do not relate to a particular mass of protein. This is done because it is difficult to know precisely the efficiency of a particular phosphorylation reaction, and therefore, the ratio of phosphorylated to nonphosphorylated protein in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphoELISA, and are described within the protocol booklet that comes with the assay.

For example, a typical unit description would be "1 unit = the amount of AKT[pS473] derived from 100 pg of AKT phosphorylated by MAPKAP2 and PDK1" as is seen for our AKT[pS473] phosphoELISA assay. Since there is no guarantee that the AKT in our standard prep is 100% phosphorylated, we refrain from making the statement that this corresponds to 100 pg of phosphorylated AKT. Instead, we validate a large batch of phosphorylated protein, and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to insure that our unit definitions remain constant from lot to lot. In order to evaluate phosphorylation levels, we report comparitive levels of phosphorylation of a protein, in units of phosphoprotein per picogram or nanogram of total protein. The total ELISA kit quantifies the mass of protein per sample and the phosphoELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) increase, decrease, or remain constant between various samples.

Example determination: Two samples tested for CREB [pS133]:
Sample 1 results:
CREB (Total) Human ELISA Kit results show 100 pg/mL CREB in the sample. phosphoELISA CREB [pS133] Human ELISA Kit results show 50 units/mL of CREB phosphorylated at serine 133. For this sample, CREB is phosphorylated at serine 133 to the level of (50 units/mL)/(100 pg/mL) = 0.5 units/pg of CREB protein.
Sample 2 results:
CREB (Total) Human ELISA Kit results show 95 pg/mL CREB in the sample. phosphoELISA CREB [pS133] Human ELISA Kit results show 5 Units/mL of CREB phosphorylated at serine 133. For this sample. CREB is phosphorylated at serine 133 to the level of (5 units/ml)/(95 pg/ml) = 0.05 units/pg of CREB protein.

Answer Id: E5163

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Product FAQ

How do I assess the phosphorylation state of a protein using a BioSource phosphoELISA kit?

Answer

Our phosphoELISA assays are designed to quantify the phosphorylation states of various proteins involved in signal transduction. Our method involves the use of two sandwich ELISA assays: phospho specific ELISA and total ELISA. Both ELISA assays capture total protein, regardless of phosphorylation state, to the wells of an ELISA plate. This is done by coating the plates with a “pan” antibody that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein, and does not block the phosphorylation site to be studied. Total ELISA then quantifies the total amount of protein present in the sample by using a second “pan” antibody for detection of protein regardless of phosphorylation state. This measurement is given in picograms/mL of sample, or sometimes nanograms of protein per mL. The measurement is always given in mass units. This is because standards of known mass are available for use in the standard curve for the total assay.

Phospho specific ELISA quantifies the amount of that same protein that has been phosphorylated at a specific amino acid. Instead of a second “pan” antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated at a particular amino acid position. This procedure is extremely similar to an immunoprecipitation reaction in which two different antibodies are used; one to capture a protein from a solution and one to detect the subset of that protein population that is phosphorylated at a certain site. However, the ELISA assay has several advantages over immunoprecipitation, including ease of use and increased sensitivity. ELISA is also quantitative. This phospho specific ELISA assay will detect and quantify only the phosphorylated form of a particular protein, and its measurement is given in “units” which we do not relate to a particular mass of protein. This is done because it is difficult to know precisely the efficiency of a particular phosphorylation reaction, and therefore the ratio of phosphorylated to nonphosphorylated protein in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phospho specific ELISA, and are described within the protocol booklet that comes with the assay. For example, a typical unit description would be “1 unit = the amount of AKT[pS473] derived from 100 pg of AKT phosphorylated by MAPKAP2 and PDK1” as is seen for our AKT[pS473] phosphoELISA assay. Since there is no guarantee that the AKT in our standard prep is 100% phosphorylated, we refrain from making the statement that this corresponds to 100 pg of phosphorylated AKT. Instead, we validate a large batch of phosphorylated protein and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to insure that our unit definitions remain constant from lot to lot.

In order to evaluate phosphorylation levels, we report comparative levels of phosphorylation of a protein, in units of phosphoprotein per picogram or nanogram of total protein. The total ELISA kit quantifies the mass of protein per sample and the phospho specific ELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) increase, decrease, or remain constant between various samples.

Example determination: Two samples tested for CREB [pS133]:
Sample 1 results: Total assay (Cat. No. KHO0231) shows 100 pg/mL CREB in the sample. Phospho specific ELISA (Cat. No. KHO0241) shows 50 units/mL of CREB phosphorylated at serine 133. For this sample, CREB is phosphorylated at serine 133 to the level of (50 units/mL)/(100 pg/mL) = 0.5 units/pg of CREB protein.
Sample 2 results: Total assay results show 95 pg/mL CREB in the sample. Phospho specific ELISA results show 5 units/mL of CREB phosphorylated at serine 133. For this sample, CREB is phosphorylated at serine 133 to the level of (5 units/mL)/(95 pg/mL) = 0.05 units/pg of CREB protein. Then we can compare Samples 1 and 2 for the serine 133 phosphorylation level change.

Answer Id: E5141

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Product FAQ

What units of measurement do you use for the results of total and phosphospecific ELISAs?

Answer

The results of our total ELISAs are given in pg/mL of sample, or sometimes ng/mL. This measurement is always given in mass units because standards of known mass are used to prepare the standard curve. The results of the phosphospecific ELISAs are given in “units”, which we do not relate to a particular mass of protein. We use units because it is difficult to precisely know the efficiency of a particular phosphorylation reaction, and therefore the ratio of phosphorylated to unphosphorylated protein, in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphospecific ELISA and are described within the product manual that accompanies each kit.

For example, a typical unit description would be “1 unit = the amount of FAK [pY397] derived from 300 pg of auto-phosphorylated FAK protein”. Since there is no guarantee that the FAK in our standard preparation is 100% phosphorylated, we refrain from making the statement that this corresponds to 300 pg of phosphorylated FAK. Instead, we validate a large batch of phosphorylated protein and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to ensure that our unit definitions remain constant from lot to lot.

Answer Id: E12622

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Manual / Product Insert

ELISA Technical Guide

Version: 10 April 2012
Catalog #

Manual / Product Insert

User Guide: Phosphoprotein Phosphate Estimation Assay Kit

Version: Jan. 2015
Catalog #

Manual / Product Insert

PeppermintStick Phosphoprotein Molecular Weight Standards

Version: P33362 07-16-2004
Catalog #

Product FAQ

Can I use other molecular weight standards with the Pro-Q Diamond Phosphoprotein stain?

Answer

Other known phosphoproteins can be used as positive control standards for the Pro-Q Diamond Phosphoprotein stain. Ovalbumin, in the Protein Molecular Weight Standards Reagent (Cat. No. P6649) is a phosphoprotein. None of the proteins in the Mark12, Invitrogen Sharp, SeeBlue or SeeBlue Plus2 standards is a phosphoprotein that could be used as a positive control with the Pro-Q Diamond Phosphoprotein stain.

Answer Id: E11199

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Product FAQ

What are the characteristics of the proteins included in the PeppermintStick Phosphoprotein Molecular Weight Standards?

Answer

All proteins are single chain, monomeric proteins (no subunits). The phosphoproteins are both serine-phosphates. Ovalbumin has 2 phosphorylated residues and β-casein has 5 phosphorylated residues. We do not determine whether or not all the phosphates are present at all times for each lot and can only assume that a majority of each protein has all sites phosphorylated. Any protein extracted from a natural source may always have the potential for some dephosphorylation during extraction/purification or by natural cellular activity. For a more definitive phosphorylation number comparison to staining intensity, a synthesized peptide of known number of phosphates must be used. Please see below for details:

- β-galactosidase: 116,250 kDa; No phosphoprotein; No glycoprotein; Source - E. coli
- Bovine serum albumin: 66,200 kDa; No phosphoprotein; No glycoprotein; Source - Bovine serum
- Ovalbumin: 45,000 kDa; phosphoprotein present; glycoprotein present; Source - Chicken egg
- β-casein: 23,600 kDa; phosphoprotein present; No glycoprotein; Source - Bovine milk
- Avidin: 18,000 kDa; No phosphoprotein; glycoprotein present; Source - Chicken egg
- Lysozyme: 14,400 kDa; No phosphoprotein; No glycoprotein; Source - Chicken egg

Answer Id: E11732

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Product FAQ

How should the stock solution of PeppermintStick Phosphoprotein Molecular Weight Standards be stored?

Answer

For long-term storage, store the stock tube at either -20 degrees C or -80 degrees C. For short-term storage, store at 2-6 degrees C. The stock can be subjected to repeated freezing and thawing without any deleterious effects to the molecular weights of the proteins or their migration during electrophoresis.

Answer Id: E11731

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Product FAQ

How much protein is in the stock solution of PeppermintStick Phosphoprotein Molecular Weight Standards?

Answer

Each of the six proteins is present at approximately 0.5 µg/µL concentration.

Answer Id: E11730

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Spectra Data

Pro-Q Diamond

View data points for this Spectra or view in Fluorescence SpectraViewer.

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Spectra Data

SYPRO Ruby protein gel stain

View data points for this Spectra or view in Fluorescence SpectraViewer.

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Product FAQ

What is the sensitivity of Krypton Protein Stain?

Answer

The stain is sensitive down to 0.25 ng using the standard protocol, or down to 2 ng using the rapid 30 minute protocol. A broad range of proteins have been tested with molecular weights from 14.4 to 200 K, including glycoproteins and phosphoproteins. Some of the proteins tested are as follows: myosin, beta-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme, aprotinin, horseradish peroxidase, myokinase, avidin, glucose oxidase, a2-macroglobulin, a1-acid glycoprotein, and beta-casein. (HeLa cell and rat heart tissue lysates were tested with 2D applications).

Answer Id: E8369

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Manual / Product Insert

Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit

Version: 01-24-2006
Catalog #
  • P33706(Discontinued)

Manual / Product Insert

Photographic Filters for Fluorescent Dye–Stained Gels and Blots

Version: S32718 06-19-2007
Catalog #