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查看更多产品信息 c-Met [pYpYpY1230/34/35] Human ELISA Kit - FAQs (KHO0281)
1 个常见问题解答
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.
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