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Protein Phosphorylation Modification

Protein phosphorylation modification refers to the transfer of the phosphate group of ATP to the amino acid side chain of a protein under the catalysis of kinases. This process is generally reversible, and the protein phosphorylation modification can be removed by phosphatases. Protein phosphorylation modification usually occurs on serine, threonine, and tyrosine residues. Such modification can alter the structure and activity of proteins, thereby affecting cellular physiological processes such as metabolism, proliferation, and differentiation. It is the most basic, common, and important mechanism for regulating and controlling protein vitality and function. Current research involving protein phosphorylation includes disease mechanism research, cellular signal transduction research, drug development, developmental biology, stress response, and other fields.

Why Is Phosphorylated Protein WB Challenging?

1.       The abundance of phosphorylated proteins is usually low, accounting for only about 10% of the total protein, or even lower;

2.       Protein phosphorylation modification is reversible. Once cells are lysed, proteases and phosphatases are immediately released, leading to dephosphorylation of phosphorylated proteins within milliseconds.

Key Considerations for Phosphorylated Protein WB

1. Pre-Experiment Preparation — Fully Understand Your Research Object

Generally, review the literature or use databases to understand the phosphorylation sites and levels of the target protein. We recommend using PhosphoSitePlus — a comprehensive predictive database for post-translational modifications of proteins. Simply enter the protein name to query various modification types, site information, and other relevant data of the protein. For proteins with low expression levels, physical or chemical methods can be used for stimulation and induction.

2. Sample Processing — Take the First Step Correctly

1.       Use fresh samples;

2.       Operate quickly;

3.       Pre-cool reagents and equipment, and perform operations on ice;

4.       Avoid using ultrasonic cell disruptors for cell lysis as much as possible. For tissue grinding, pre-cool the mortar with liquid nitrogen in advance;

5.       The lysis buffer must contain both protease inhibitors and phosphatase inhibitors — neither can be omitted;

6.       Note that some phosphorylated proteins should not be boiled, as boiling may destroy their phosphorylation sites;

7.       After determining the protein concentration, add loading buffer, aliquot, and store at -80°C to avoid repeated freeze-thaw cycles.

3. Internal Reference Selection

Two internal references are required: one is the total protein corresponding to the phosphorylated protein. Detecting changes in the expression level of phosphorylated protein alone is not sufficient to draw conclusions; only changes in the ratio of phosphorylated protein to total protein can indicate changes in phosphorylation levels. The other is a general internal reference, which serves as a control for the experimental system. However, errors are relatively large when comparing samples loaded in different wells. A commonly used method is to use a primary and secondary antibody stripping solution to remove the previously bound phosphorylated antibody and secondary antibody, then probe the same membrane for total protein. Strip again and probe for the internal standard.

4. Sample Loading — Sufficient Quantity

Compared with ordinary WB, the phosphorylation level of phosphorylated proteins is lower, so a sufficient amount of sample should be loaded.

5. Blocking — Choose the Right Blocking Agent

Milk contains casein, a phosphoprotein that can cause high background. For the detection of phosphorylated proteins, it is recommended to use bovine serum albumin (BSA) or protein-free blocking agents. Of course, if the antibody instructions specify a recommended blocking agent, follow the instructions.

6. Antibody Incubation

For primary antibodies against phosphorylated proteins, it is best to incubate overnight at 4°C to ensure sufficient binding time. Incubation at 4°C overnight also prevents the protein antigen from falling off the membrane. Secondary antibodies can be incubated at room temperature for 1 hour.

7. Washing — Attention to Details

For initial experiments, it is recommended to reduce the number of washing steps and the rotational speed of the shaker. Having some background is better than having no signal at all. Later, you can optimize appropriately to obtain ideal results.

References

Pan B, Zhu X, Han B, Weng J, Wang Y, Liu Y. The SIK1/CRTC2/CREB1 and TWIST1/PI3K/Akt/GSK3β signaling pathways mediated by microRNA-25-3p are altered in the schizophrenic rat brain. Front Cell Neurosci. 2023 Jan 20;17:1087335. doi: 10.3389/fncel.2023.1087335. PMID: 36744005; PMCID: PMC9896578.

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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 challenges in phosphorylated protein WB experiments and the urgent need for standardized, specialized experimental tools. Through our specialized sub-brands (Absin, Starter, UA), we have developed a targeted product portfolio for phosphorylated protein WB, covering core phosphorylated antibodies, sample processing reagents (lysis buffers, protease/phosphatase inhibitors), protein quantification kits, WB experimental reagents, and protein markers.

Our team adheres to stringent quality control standards throughout the product development and production process, ensuring the high specificity, sensitivity, and reproducibility of each product. We are committed to providing professional technical support and customer-centric services, helping researchers overcome experimental challenges such as low abundance of phosphorylated proteins, easy dephosphorylation, and high background, and accelerating the pace of scientific research breakthroughs in related fields. ANT BIO PTE. LTD. strives to be a trusted partner for scientists worldwide, contributing to the advancement of protein phosphorylation research and the development of innovative biological technologies.

Disclaimer

This article is AI-compiled and interpreted based on the original work related to phosphorylated protein WB experiment 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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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 phosphorylated protein detection antibodies and sample processing reagents to WB experimental reagents and protein markers. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in overcoming phosphorylated protein WB challenges and driving biological technology progress. Explore our product portfolio today and elevate your research to new heights.