Testing of Total Protein or Phosphorylated Protein

Phosphorylation evaluation

Protein kinases transfer phosphate groups from adenosine triphosphate to serine, threonine or tyrosine residues on protein peptide substrates, which directly affect the activity and function of targets. Radioactive studies have shown that about 30% of proteins in eukaryotic cells have been phosphorylated. This key post-translational modification regulates a wide range of cellular activities, including cell cycle, differentiation, metabolism and neuronal communication. In addition, abnormal phosphorylation events are associated with many disease states. When evaluating phosphorylation, the method selected may vary, depending on a number of factors, including the specific issues raised and the availability of special instruments or reagents. How to detect protein phosphorylation, this paper briefly introduces several common methods, and puts forward the advantages and disadvantages of each method.

Kinase activity analysis

Protein kinases are usually common components of multiple signal transduction networks, which affect many downstream effectors responsible for biological reactions. Therefore, evaluating the activity of a specific kinase may provide valuable information for parallel pathways. Kinase activity in biological samples is usually measured in vitro. Immunoprecipitated kinases are incubated with exogenous substrates in the presence of adenosine triphosphate. The phosphorylation of substrates by specific kinases was then evaluated by a number of reporting systems, including chromogenic, radioactive or fluorescent detection.

In addition, the R & D system also provides a non radioactive universal kinase activity kit, which can quantify the activity of any kinase that can produce ADP. Although we can obtain information about the behavior of specific kinases, evaluating the enzyme activity in cell extracts only reveals the tip of the iceberg of signaling pathways. Little is known about how proteins are modified, and in vitro activity analysis can not explain the potential role of endogenous phosphatase activity. The direct detection of phosphorylated proteins may provide a more detailed analysis of how cells respond to external stimuli, because the identification of phosphorylated peptides provides information for protein expression and functional status.

Development of phosphorylation specific antibodies

A classical method for direct determination of protein phosphorylation is to incubate the whole cell with radiolabeled 32P phosphate to obtain the cell extract, which is separated by SDS-PAGE and exposed to film. This cumbersome method requires several hours of incubation and the use of radioisotopes. Other traditional methods include two-dimensional gel electrophoresis, which assumes that phosphorylation changes the protein mobility and isoelectric point.

Since these methods are laborious, the development of phosphorylation dependent antibodies has been greatly welcomed by researchers. In 1981, the first recorded phosphorylated antibody was produced in rabbits, Keyhole haemocyanin was used (Kuala Lumpur) benzoyl phosphate conjugate. This antibody widely recognized proteins containing phosphotyrosine. Ten years later, rabbits were immunized with synthetic phosphorylated peptides to develop multiple phosphorylated state specific antibodies. These phosphorylated peptides represent the amino acid sequence around the phosphorylation site of the target protein. Then, the immune serum was loaded into the peptide affinity column, Produce highly specific immune reagents. The emergence of phosphorylated specific antibodies has opened the door for the improvement of traditional methods and the development of new immunoassay techniques. The advice for using phosphorylated specific antibodies in any technology is that successful detection depends on the specificity and affinity of the antibody to the phosphorylated protein of interest.

western blot

Western blotting is the most commonly used method to evaluate protein phosphorylation. Most cell biology laboratories have the equipment to carry out these experiments. The biological samples were separated by SDS-PAGE, then transferred to the membrane (usually polyvinylidene fluoride or nylon membrane), and then the target protein was identified by phosphorylation specific antibody.

The typical Western blot procedure avoids the requirements of hazardous materials and waste disposal when radioisotopes are used. Many phosphorylation specific antibodies are very sensitive and can easily detect phosphorylated proteins in conventional samples (such as 10-30 micrograms of cell extract). Because the measured level of phosphorylated protein may vary with the processing or gel sample error, researchers often use an antibody to detect the total level of homologous protein (without considering the phosphorylation state), to determine the proportion of phosphorylation components relative to the total components, and to act as a sample control. Chemiluminescence and chromogenic methods are commonly used, and molecular weight markers are often used to provide information on protein molecular weight. See www.rndsystems for detailed steps of Western blotting. com/go/WesternBlotProtocol

Enzyme linked immunosorbent assay (ELISA)

Enzyme linked immunosorbent assay has gradually become a powerful method for the determination of protein phosphorylation. The quantitative ability of enzyme-linked immunosorbent assay is better than Western blot, and it plays a great role in the study of regulating kinase activity and function. This kind of microplate analysis generally uses the capture antibody specific to the target protein, which is independent of the phosphorylation state. Then the target protein is bound to the antibody coated analysis plate. The target protein can be pure or a component of complex heterogeneous samples (such as cell lysate). Add the detection antibody specific to the phosphorylation site to be analyzed. These analyses are usually designed for chromogenic or fluorescent detection.

The resulting signal intensity is directly proportional to the concentration of phosphorylated protein present in the initial sample. Compared with more traditional Western blotting, phosphorylation specific enzyme-linked immunosorbent assay has some advantages. First, the results can be easily quantified using a calibrated standard. Secondly, the use of two antibodies specific for the target protein in the form of sandwich brings high specificity. Third, the higher sensitivity of ELISA allows the use of a smaller number of samples to detect low abundance proteins. Finally, the flux in the form of microplate is much higher than that of traditional Western blotting. Enzyme linked immunosorbent assay usually leads to indirect determination of kinase activity. However, another kind of enzyme-linked immunosorbent assay uses immobilized capture antibodies, substrates and phosphorylated substrates, which leads to a more direct determination of kinase activity.

Cell based enzyme linked immunosorbent assay

Although biochemical kinase analysis in vitro (such as typical sandwiches (ELISA) are commonly used in hypothesis testing and drug screening, but they cannot replicate the intracellular environment. Analyzing protein phosphorylation in intact cells may more accurately represent the state of specific signal pathway networks. Some immunoassays have recently been developed to determine protein phosphorylation in the context of intact cells. Cells are stimulated, fixed and blocked in the same well. Phosphorylation specific antibodies were used to evaluate the phosphorylation status by fluorescence or color detection system. In addition, phosphorylated protein and total protein were detected simultaneously in the same well of the microplate. Therefore, the signal from the target protein can be standardized by the second protein, correct the difference between wells, realize the accurate evaluation of phosphorylated protein level, and compare multiple samples, which is similar to the phosphorylated specific and total protein antibodies used in traditional Western blotting. These analyses bypass the need to prepare cell lysates and are therefore more suitable for high-throughput analysis.

Intracellular flow cytometry and ICC / IHC

Conventional intracellular flow cytometry and immunocytochemistry / immunohistochemistry (ICC / IHC) is a powerful tool for detecting phosphorylation events. Flow cytometry uses laser to excite fluorescent dyes for antibody detection. When evaluating multiple proteins in the same cell, filter combinations and fluorescent dyes must be carefully selected, and their spectra cannot overlap. Flow cytometry has great advantages because it enables rapid and quantitative single-cell analysis. Through the cell surface Marker typing can detect proteins of specific cell types in heterogeneous populations without physically isolating cells. This method can analyze rare cell populations without worrying about cell loss or possible changes in protein expression during cell sorting.

ICC usually refers to the detection of proteins in cultured cells by microscope, while IHC refers to the detection of proteins in complete tissue sections. Similar to flow cytometry, these techniques realize the evaluation of multiple proteins in cells or tissues, but enough attention should be paid to avoid the overlap of fluorescence spectra or colors. Fluorescence and color detection techniques are often used. Unlike other forms of monitoring phosphorylation, ICC is usually the preferred method for determining intracellular localization. Both flow cytometry and ICC / IHC require high affinity and high specificity antibodies, blocking steps, control and antibody titration to avoid unclear results due to nonspecific binding.

The detection of phosphorylated proteins by flow cytometry and ICC requires that the proteins are stable and accessible to antibodies. Cells are usually stimulated and fixed by formaldehyde or paraformaldehyde to crosslink phosphorylated proteins to make them stable and easy to analyze. The immobilized cells must be permeabilized so that phosphorylated specific antibodies can enter the cells. For different subcellular localization, different permeabilization techniques are usually used. Mild detergents can detect cytoplasmic proteins, and alcohol may be needed for antibodies to approach nuclear proteins. Alcohol penetration can also enhance the detection of phosphorylated protein of phosphorylated specific antibody, because alcohol has the characteristics of denaturation. For more detailed ICC / IHC experimental steps, please visit www.rndsystems. com/go/IHCProtocol

mass spectrum

A comprehensive assessment of protein phosphorylation (phosphorylated proteomics) in complex biological samples (such as cell lysates) is important for understanding phosphorylation based signal networks. Large scale phosphorylated protein analysis in complex protein mixtures includes the identification of phosphorylated proteins and phosphorylated peptides, and the sequencing of phosphorylated residues. Mass spectrometry (MS) technology is a useful tool to complete these tasks.

Although mass spectrometry has excellent sensitivity and resolution in identifying individual proteins, there are some inherent difficulties in the analysis of phosphorylated proteins. First, the signals of phosphorylation peptides are usually weak because they are negatively charged, while electrospray ionization mass spectrometry acts in positive mode, so the ionization effect is not good. Secondly, under the high background of a large number of non phosphorylated proteins, it is difficult to observe the signal of low abundance target proteins. In order to overcome these shortcomings, some enrichment strategies have been developed, including immobilized metal affinity chromatography (IMAC) phosphorylation specific antibody enrichment and chemical modification (such as phosphorylation of serine and threonine) β- Elimination reaction), and replacing the phosphate group with a biotinylated group.

Multiple analyte map analysis

Mass spectrometry techniques such as collision induced dissociation (CID) and electron transfer dissociation (ETD) provide a comprehensive parallel analysis of peptide sequences and post-translational modifications (phosphorylation). These techniques are quite laborious, and a comprehensive phosphorylation analysis strategy may not be required when a specific pathway is the main research target. This has led to the development of new methods for the simultaneous determination of protein phosphorylation of multiple analytes. In general, these methods involve the use of phosphorylated specific antibodies, including microplate, magnetic bead and membrane based detection forms. The most obvious advantage of these analyses is that the flux capacity is greatly improved, avoiding the need to run multiple Western blots or traditional enzyme-linked immunosorbent assay. These techniques can also provide more data and require very few samples. Accordingly, protein profiling is generally considered less sensitive than traditional correspondence techniques due to potential antibody cross reactivity.

Phosphorylated protein multiplex

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