Through stringent R&D, our mass spectrometry-based phosphoproteomics platform integrates a variety of enrichment strategies, aiming to meet our clients' diverse experimental demands.

Antibody array

Applying advanced sandwich antibody array technology, It is capable of conducting precise multiplex detection of phosphorylated proteins. In this process, the antibodies are meticulously immobilized onto surfaces such as glass, membranes, or magnetic beads. After the blocking steps are completed, we co-incubate the sample with the array. Subsequently, non-specific proteins are washed off and the array is co-incubated with detection antibodies for chemiluminescent or fluorescent analysis. The intensity of the signal is proportionate to the quantity of the antibody-bound protein. When we juxtapose the data from western blotting, ELISA, and the antibody array, all three techniques demonstrate analogous patterns in phosphorylation changes. Although the antibody array can offer quantitative data, the current market does not yet have quantifiable chip products for phosphorylated proteins. However, by adopting a readily addressable approach, we can easily discover or label different antibodies, thereby streamlining the protein identification process.

Western blot

Western blotting is currently the preferred method for evaluating the phosphorylation status of proteins, with many cell biology laboratories equipped to perform such experiments. This method separates biological samples through SDS-PAGE and subsequently transfers them to a membrane—typically, PVDF or nitrocellulose. Following this, target proteins are identified using antibodies specific for phosphorylation. The standard process for Western blot experiments eliminates the safety risks and waste disposal requirements incurred by the usage of radioactive isotopes.

Numerous phosphorylation-specific antibodies exhibit high sensitivity, enabling the effortless detection of phosphorylated proteins in common samples-- such as cell extracts in the range of 10-30 micrograms. Given the potential impact of handling or gel-loading errors on the measurement of phosphorylated protein levels, researchers frequently use one antibody to detect the total levels of a homologous protein (irrespective of its phosphorylation state). This benchmarking process facilitates the determination of the ratio of phosphorylated components to total components and serves as an internal control for loading.

Both chemiluminescent and colorimetric methods are extensively used, while molecular weight markers assist in providing information about the protein's molecular weight.

ELISA

As an indispensable tool, Enzyme-Linked Immunosorbent Assay (ELISA) has gradually become a crucial method for measuring protein phosphorylation. In comparative terms, ELISA supersedes Western blotting in its quantitative prowess, and showcases significant potential in investigating kinase activity and function. This microplate-based analytical method primarily relies on capture antibodies that are specific to the target protein, regardless of its phosphorylation state.

In this process, the target protein binds to an antibody-coated analysis plate; this protein could be a component of purified products or complex specimens such as the lysate of cells. Following this, a detection antibody pertaining to a particular phosphorylation site is introduced. These analyses generally employ colorimetric or fluorescent detection methods, and the intensity of the resulting signal is directly proportional to the concentration of the phosphorylated protein in the original sample.

Compared to traditional immunoblotting, phosphorylation-specific ELISA showcases substantial advantages. Firstly, quantification of results is readily achieved via calibrated standards. Secondly, the application of a sandwich-style dual antibody strategy significantly amplifies specificity. In addition, the high sensitivity of ELISA permits the use of fewer samples, thus enabling the detection of low abundance proteins. Finally, the microplate format materially enhances analytical throughput, rendering it more advantageous than traditional Western blotting.

It is noteworthy that ELISA is typically used for the indirect determination of kinase activity. Nevertheless, another version of ELISA technology employing immobilized capture antibodies, substrate, and detection methods for phosphorylated substrates provides a more direct means for measuring kinase activity.

In-Cell ELISA

"In-Cell ELISA" In what has been a widespread application for hypothesis testing and drug screening, biological biochemical kinase assays such as traditional sandwich Enzyme-Linked Immunosorbent Assays (ELISA) have played an immense role. However, these assays might not faithfully mirror the intricate environments within cells. Therefore, studying protein phosphorylation processes inside intact cells may provide a more authentic reflection of the state of specific signaling pathway networks.

Recently, several immunological assays have been developed that are designed for intact cells, capable of quantifying intracellular protein phosphorylation levels. In these methods, stimulation, fixation, and blocking processes are performed within the same well. Subsequently, using the specificity of antibodies against phosphorylation sites along with either a fluorescent or colorimetric detection system, protein phosphorylation states are evaluated.

Notably, these innovative methods are equipped with the capacity to concurrently measure phosphorylated proteins and total proteins within the same well. This design permits the use of a secondary protein signal for the normalization of the target protein signal, thereby compensating inter-well variances and ensuring accurate assessment of phosphorylated protein levels. This matches traditional immunoblotting techniques, where antibodies specific for phosphorylated proteins and total protein are utilized.

As these novel analysis methods eliminate the need to prepare cell lysates, they are better suited for high-throughput analysis. By reducing operation steps and sample processing time, they equip researchers with more robust, accurate tools for protein phosphorylation studies.

Flow Cytometry and ICC/IHC

The conventional intracellular flow cytometry and immunocytochemistry/immunohistochemistry (ICC/IHC) play pivotal roles in the detection of phosphorylation events. Flow cytometry exploits lasers to excite fluorescent dyes for antibody detection. When evaluating multiple proteins within a single cell, the judicious selection of filter combinations and fluorescent dyes is crucial to ensure their spectra do not overlap. The substantial advantage of flow cytometry lies in its capability to conduct rapid and quantitative single-cell analyses. By identifying cell surface markers, this technique can detect proteins in distinct cell types within heterogeneous populations without necessitating physical separation. This methodology is particularly applicable to the analyses of rare cell populations and effectively circumvents issues arising from cell loss or protein expression alterations during cell sorting processes.

Meanwhile, ICC generally refers to the microscopic examination of proteins within cultured cells, while IHC is intended for proteins within intact tissue sections. These techniques, akin to flow cytometry, can assess multiple proteins within cells or tissues, albeit with special attention required to avoid overlapping fluorescent spectra or colors. Both fluorescence and chromogenic detection techniques are broadly utilized in such applications. Compared to other means of monitoring phosphorylation, ICC is typically the preferred method for determining intracellular localization. Whether it's flow cytometry or ICC/IHC, the use of highly affinity-specific antibodies is required, together with the implementation of blocking steps, controls, and antibody titration measures, to obliterate potential interferences stemming from non-specific binding.

The use of flow cytometry and ICC for detecting phosphorylated proteins is predicated on the prerequisite that the protein remains stable, and the antibody can approach its target. Cells typically need to be stimulated and fixed via formaldehyde or paraformaldehyde to stabilize and cross-link phosphorylated proteins for subsequent analyses. Once fixed, cells also need to undergo a permeabilization process to allow access to phosphorylation-specific antibodies inside the cells. Different permeabilization techniques are required for different subcellular localizations. Mild detergents are suitable for cytoplasmic protein detection, whereas alcohol or other permeabilization agents might be necessary for nuclear protein detection. Alcohol permeabilization not only enhances the detection efficiency of phosphorylation-specific antibodies towards phosphorylated proteins but also offers unique advantages due to its denaturing properties.

Multiple Parallel Analysis through Mass Spectrometry

Mass spectrometry techniques, such as Collision-Induced Dissociation (CID) and Electron Transfer Dissociation (ETD), offer expansive parallel analytic capabilities in contemporary studies of peptide sequencing and post-translational modifications, particularly phosphorylation. Despite these approaches' high-depth analysis, comprehensive phosphorylation analysis strategies might not always be imperative when focused on specific pathway investigations. Consequently, a myriad of novel methods have been developed for simultaneous assessment of the phosphorylation state of multiple analytes.

These techniques primarily rely on the application of phospho-specific antibodies, encompassing detection platforms based on microplates, magnetic beads, and membranes. A considerable advantage that these newly-emerged methods possess is their substantial amplification of throughput capacity, thus curtailing the necessity for multiple Western blot or traditional enzyme-linked immunosorbent assay (ELISA) analyses. Furthermore, these methodologies can provide an abundance of data, even with minimal sample volumes.

Notably, however, proteomic analytic techniques generally exhibit lower sensitivity compared to their conventional counterparts, predominantly due to potential antibody cross-reactivity. Therefore, consideration of these methods' limitations is required, in conjunction with thoughtful selection and application relative to specific research objectives and conditions.