Glass Gel Phosphoprotein Precast Gel: Principles, Technologies, and Applications

 I. Background Introduction: Protein Phosphorylation

Protein phosphorylation is a crucial post-translational modification (PTM) that involves the addition of phosphate groups to specific amino acid residues of proteins. This process is typically catalyzed by a class of enzymes called protein kinases, while the removal of phosphate groups is catalyzed by phosphatases. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues.

Figure 1 Schematic Diagram of Protein Phosphorylation

1. Common Protein Kinases

The International Union of Biochemistry classifies protein kinases into the following categories based on the specificity of acceptor amino acids:

1.       Phosphotransferases that use protein hydroxyl groups as acceptors are called protein serine or threonine kinases;

2.       Phosphotransferases that use phenyl groups as phosphate acceptors are called protein tyrosine kinases;

3.       Phosphotransferases that use His, Arg, or Lys as acceptors are called protein His kinases;

4.       Phosphotransferases that use Cys residues as acceptors are called protein Cys kinases;

5.       Phosphotransferases that use acetyl groups as acceptors are called aspartate or glutamine kinases.

The first two types of enzymes are the most common, and many protein serine/threonine or tyrosine kinases have been purified.

2. Significance of Protein Phosphorylation Research

1.       Cellular Signal Transduction and Regulation: Protein phosphorylation plays a key role in intracellular signal transduction. Through phosphorylation modification, proteins can be activated or inhibited, thereby participating in the regulation of various intracellular signaling pathways, including cell proliferation, apoptosis, differentiation, etc. This modification method exerts important effects in biological processes such as cellular signal transduction, gene expression regulation, and cell cycle regulation;

2.       Cell Cycle and Proliferation: Protein phosphorylation plays an important role in various stages of the cell cycle. Phosphorylation modification can regulate the activity of cyclins, control the orderly division and proliferation of cells, and maintain the normal function of cells;

3.       Disease Occurrence and Treatment: The occurrence of many diseases is related to abnormalities in protein phosphorylation. Studying changes in protein phosphorylation can help us understand the pathogenesis of diseases, discover potential therapeutic targets, and develop new drug treatment strategies. Abnormal protein phosphorylation is associated with many diseases, especially cancer. For example, protein kinases encoded by certain oncogenes may lead to inappropriate phosphorylation, thereby promoting tumorigenesis and development;

4.       Drug Development and Screening: By analyzing changes in protein phosphorylation, new drug targets and signaling pathways can be discovered, guiding drug design and development. At the same time, protein phosphorylation analysis can also be used for drug screening to evaluate the regulatory effect of drugs on specific phosphorylation sites;

5.       Personalized Medicine: Protein phosphorylation analysis can provide important guidance for personalized medicine. By detecting the status of phosphorylation sites, the patient's response to specific treatments can be predicted, providing a basis for clinical treatment decisions;

6.       Biomarker Identification: Researchers have discovered many disease-related biomarkers by analyzing changes in protein phosphorylation. These biomarkers can be used for early disease diagnosis, prognosis evaluation, and treatment monitoring;

7.       Protein Function and Stability: Phosphorylation can change the conformation of proteins, thereby altering their activity and/or stability. For example, phosphorylation can regulate enzyme activity by changing the active state of enzymes. In addition, phosphorylation can also regulate protein stability by changing the degradation rate of proteins;

8.       Protein-Protein Interactions: Phosphorylation can change the interactions between proteins or between proteins and other molecules. For example, phosphorylation can mediate the binding of proteins to ligands, thereby affecting the activation of signal transduction pathways. In addition, phosphorylation can also change the affinity or reactivity between proteins, thereby affecting complex network interactions in cells.

II. Common Detection Methods for Protein Phosphorylation

1.       Western Blotting: This is a commonly used protein detection technology that can be used to detect protein phosphorylation levels. Key steps include protein extraction, protein separation and transfer, antibody recognition, and signal detection. By selecting specific phospho-specific antibodies, the phosphorylation level of the target protein can be accurately detected;

2.       Mass Spectrometry (MS): Mass spectrometry is a high-sensitivity and high-resolution protein analysis technology that can be used to detect protein phosphorylation sites and quantify phosphorylation levels. Commonly used methods include liquid chromatography-tandem mass spectrometry (LC-MS/MS) and phosphopeptide enrichment. By analyzing mass spectrometry data, phosphopeptides can be identified and quantified, and phosphorylation sites can be determined;

3.       Enzyme-Linked Immunosorbent Assay (ELISA): Phospho-specific antibodies can be used to quantitatively analyze the phosphorylation level in protein samples. This microplate-based assay generally uses a capture antibody specific for the target protein, regardless of its phosphorylation state. Subsequently, the target protein is bound to the antibody-coated assay plate, and a detection antibody specific for the phosphorylation site to be analyzed is added;

4.       Radioisotope Labeling: By using 32P isotope-labeled phosphate as the phosphate group donor, through phosphorylase-catalyzed reactions, the 32P isotope-labeled phosphate group is transferred to the corresponding reaction protein. After separation by gel electrophoresis, phosphorylated proteins can be detected by autoradiography or phosphoimaging screens.

Immunofluorescence and flow cytometry are also tools for protein phosphorylation research. In addition, a new technology for protein phosphorylation detection is introduced today—Phos-iso SDS-PAGE.

III. Phos-iso SDS-PAGE Technology

1. What is Phos-iso SDS-PAGE?

This is a phosphate affinity electrophoresis technology that can separate phosphorylated and non-phosphorylated proteins using conventional SDS-PAGE procedures. The core component of this technology, Phos-iso Acrylamide, is a binuclear metal complex that can specifically bind to phosphate groups, enabling the separation and detection of phosphorylated proteins. During use, it is only necessary to add Phos-iso Acrylamide and metal ions (Mn2+ or Zn2+) to the conventional SDS-PAGE gel. During electrophoresis, the Phos-iso complex immobilized on the gel can specifically bind to phosphorylated proteins, reducing the electrophoretic mobility of phosphorylated proteins, thereby achieving the separation of phosphorylated and non-phosphorylated proteins. Phos-iso SDS-PAGE is easy to operate, can detect different forms of phosphorylated proteins, is not limited by phospho-specific antibodies, and can be used for Western blotting, mass spectrometry, etc.

2. Technical Advantages of Phos-iso SDS-PAGE

1.       No need to prepare antibodies against phosphorylated proteins; total antibodies instead of anti-phosphorylated protein antibodies can be used to detect both phosphorylated and non-phosphorylated proteins simultaneously;

2.       Suitable for subsequent operations such as Western blotting and mass spectrometry;

3.       Phosphorylated forms with different numbers and positions of phosphorylation sites can also be separated.

IV. New Product Launch from ANT BIO PTE. LTD. – Glass Gel Phosphoprotein Precast Gel

Glass gel phosphoprotein precast gel (matched with abs9941 Phosphoprotein Loading Buffer (3×)) is pre-added with 100μmol/L Phos-iso Acrylamide and can be used directly after opening the package. The precast gel contains zinc as the metal ion, which has good stability when stored in the central gel buffer, and the resulting bands are neat. After separation, the gel can be used for Coomassie brilliant blue staining, Western blotting, and mass spectrometry experiments.

Product Features

1.       Adopts automated gel casting production technology to ensure high stability and reproducibility of product quality;

2.       Uses glass gel plates to effectively reduce non-specific protein adsorption, making protein bands more sharp and clear;

3.       The gel clamp can be opened extremely easily, only need to gently scratch one side of the gel clamp with a blade;

4.       Compatible with mainstream mini electrophoresis tanks on the market, such as Bio-Rad, Invitrogen, Tanon, and Junyi Dongfang, etc.

Figure 3 Phosphoprotein Electrophoresis Example Diagram (+: Phosphorylated sample; -: Dephosphorylated sample)

V. Frequently Asked Questions (FAQs)

1.       Band Curvature
       

a.      The sample contains inorganic salts, surfactants, EDTA, vanadic acid, etc. (samples can be treated by TCA precipitation or dialysis desalting);

b.      Improper sample handling, such as viscous and acidic samples (denature sufficiently; if the acidic sample solution is yellow-orange, add Tris buffer to neutralize to blue-purple);

c.       The pre-stained Marker band is curved due to chelating agents, leading to curvature of adjacent bands; blank lanes can also cause band curvature (use loading buffer to separate samples and Marker);

d.      High electrophoresis temperature.

2.       Unable to Distinguish Phosphorylated Bands
        Perform conventional SDS-PAGE to verify that no band migration has occurred.

3.       How to Choose Between Phospho-Specific Antibodies and Phos-iso SDS-PAGE?
        If using phospho-specific antibodies, it is necessary to strip the antibody from the membrane after incubating with the phospho-specific antibody, and then incubate with the total protein antibody. If using Phos-iso SDS-PAGE, phosphorylated (or several different phosphorylated forms) and non-phosphorylated bands can be marked on the same membrane, and the amounts of the two can be compared according to the band gray scale.

4.       Must the Sample be Purified Protein?
        Not necessarily; cell lysates can also be used. Purified samples can be detected by Phos-iso SDS-PAGE directly. For cell lysates, Western Blotting is required.

VI. Precautions

1.       Do not store below 0°C. The gel will freeze below 0°C, producing bubbles and cracks, leading to gel scrapping;

2.       Sample pretreatment is required, and the quality of the sample greatly affects the electrophoresis effect;

3.       Electrophoresis speed will slow down;

4.       Molecular weight cannot be inferred from molecular weight markers;

5.       EDTA treatment is required for membrane transfer;

6.       Conventional SDS-PAGE should also be performed simultaneously;

7.       Phosphoprotein precast gel SDS-PAGE is very sensitive to impurities in protein samples, especially chelating agents, vanadic acid, inorganic substances, and surfactants. It is strongly recommended to reduce the impurity content by TCA precipitation or dialysis before phosphoprotein precast gel SDS-PAGE;

8.       For your safety and health, please wear a lab coat and disposable gloves during operation.

VII. Product Recommendation of ANT BIO PTE. LTD.

Brand Mission

ANT BIO PTE. LTD. is dedicated to advancing life science research by providing high-quality, reliable reagents and comprehensive solutions. We recognize the critical role of efficient and accurate protein phosphorylation detection in downstream experiments such as signal transduction research, disease mechanism exploration, and drug development, and the urgent need for standardized, specialized detection tools. Through our specialized sub-brands (Absin, Starter, UA), we have developed a full-spectrum product portfolio tailored to diverse protein phosphorylation research needs, including Glass gel phosphoprotein precast gels, supporting buffers, and staining kits.

Our team adheres to stringent quality control standards throughout the product development and production process, ensuring the consistency, stability, and reliability of our products. We are committed to providing professional technical support and customer-centric services, helping researchers overcome experimental challenges such as unclear bands, poor reproducibility, and sample impurity interference, and accelerating the pace of scientific research breakthroughs. ANT BIO PTE. LTD. strives to be a trusted partner for scientists worldwide, contributing to the advancement of life science research and the development of innovative therapeutic strategies.

Disclaimer

This article is AI-compiled and interpreted based on the original work related to protein phosphorylation research. All intellectual property (e.g., experimental protocols, data, images) of the original publication shall belong to the relevant research team. For any infringement, please contact us promptly and we will take immediate action.

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ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from phosphoprotein detection reagents and precast gels to general kits and antibodies. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.