The application of L-arginine in electrophoresis

As a basic amino acid (with a guanidine group in its side chain and an isoelectric point (pI) of approximately 10.76), L-arginine, leveraging its unique chemical properties (such as charge, solubility, and protective effects on biomolecules), finds multi-scenario applications in electrophoresis technology. These applications mainly focus on three directions: biomolecule separation and purification, electrophoresis system optimization, and special function assistance. The specific application forms are as follows:

I. As an Additive in Protein/Peptide Electrophoresis to Enhance Separation Efficiency and Sample Stability

In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), native polyacrylamide gel electrophoresis (native-PAGE), or isoelectric focusing (IEF), L-arginine is often added to the gel matrix or electrophoresis buffer to improve separation performance by regulating the microenvironment of the system:

Inhibiting Protein Aggregation and Precipitation

Some proteins (e.g., membrane proteins, hydrophobic proteins, highly basic proteins) tend to aggregate due to hydrophobic interactions during electrophoresis, or precipitate when the pH environment is close to their isoelectric point, resulting in blurred bands and tailing. The guanidine group of L-arginine can bind to the hydrophobic groups of proteins, disrupting aggregation forces; meanwhile, its basic side chain maintains the local system pH away from the protein’s pI, reducing precipitation. This is particularly suitable for the electrophoretic separation of insoluble proteins such as membrane proteins and antibody fragments. For example, in native-PAGE of membrane proteins, adding 50–200 mmol/L L-arginine to the buffer can significantly reduce the protein aggregation rate and make the bands clearer.

Regulating pH and Ionic Strength of the Electrophoresis System

The basic nature of L-arginine can fine-tune the buffer pH (especially in the neutral to weakly basic range), optimizing the charged state of proteins and avoiding separation deviations caused by pH fluctuations. Additionally, as a neutral ion, it can moderately increase the buffer’s ionic strength, reducing non-specific adsorption between proteins and the gel matrix and improving separation efficiency. In IEF, if the target protein’s pI is close to the basic end (e.g., pI 9–11), adding L-arginine can help stabilize the pH gradient in the basic region and prevent the "band drifting" phenomenon during protein focusing.

Protecting Protein Activity (in Native Electrophoresis Scenarios)

In native-PAGE, proteins need to maintain their natural conformation and activity. L-arginine can reduce protein denaturation (e.g., oxidation, conformational damage) during electrophoresis through weak interactions with amino and carboxyl groups on the protein surface. This is especially applicable to the separation of environmentally sensitive biomolecules such as enzymes and active peptides, and the target protein can be directly detected by activity staining afterward.

II. Application in Capillary Electrophoresis (CE) to Improve Separation Selectivity of Small Molecules and Biomacromolecules

Capillary electrophoresis achieves separation based on differences in the mobility of molecules in an electric field. L-arginine can optimize CE separation efficiency through multiple mechanisms, particularly for polar small molecules and biomacromolecules:

As a Capillary Inner Wall Coating Modifier to Reduce Adsorption

The inner wall of the capillary is negatively charged (due to ionization of silanol groups), which easily adsorbs positively charged molecules (e.g., basic proteins, peptides), leading to peak broadening and tailing. When L-arginine is modified on the inner wall of the capillary via covalent binding or dynamic adsorption, its guanidine group can neutralize the negative charge on the inner wall and form steric hindrance, reducing electrostatic adsorption between target molecules and the inner wall. For example, in the CE separation of basic peptides, after rinsing the capillary with a 10 mmol/L L-arginine solution, the peptide peak tailing factor can be reduced from 1.8 to below 1.2, and the resolution can be improved by more than 30%.

As a Component of Background Electrolyte (BGE) to Regulate Migration Behavior

In the CE separation of small molecules (e.g., amino acids, organic acids, pharmaceutical intermediates), adding L-arginine to BGE (such as phosphate buffer or borax buffer) allows its charged groups to interact with target molecules through electrostatic or hydrogen bonding, altering the effective mobility of target molecules and thus achieving the separation of difficult-to-separate substance pairs. For example, when separating structurally similar dipeptides, adding 20 mmol/L L-arginine to BGE can expand the migration time difference between the two via hydrogen bonding between the guanidine group and the carboxyl group of the dipeptides, increasing the resolution from 1.0 (without addition) to 1.5 (meeting the requirements for baseline separation).

Assisting CE Separation of Proteins/Nucleic Acids

For CE analysis of high-purity proteins (e.g., recombinant protein purity detection), L-arginine can inhibit protein aggregation in the capillary and reduce non-specific interactions with the tube wall, resulting in more symmetric peaks. In the CE separation of nucleic acids (e.g., short-chain RNA, DNA fragments), L-arginine can stabilize the secondary structure of nucleic acids through weak electrostatic interactions, avoiding mobility fluctuations caused by structural changes and improving the repeatability of detection.

III. Assisting Separation and Detection of Basic Proteins in Two-Dimensional Electrophoresis (2-DE)

Two-dimensional electrophoresis (first dimension: IEF; second dimension: SDS-PAGE) is a core technology for separating complex protein mixtures in proteomics. However, basic proteins (pI > 8.0) are prone to loss in traditional IEF due to incomplete focusing and adsorption to gel strips. L-arginine can specifically address this issue:

Optimizing Equilibration and Focusing of First-Dimension IEF Gel Strips

Adding L-arginine (usually at a concentration of 50–150 mmol/L) to the rehydration buffer or equilibration buffer of IEF gel strips can improve the solubility of the basic region in the gel strips, reducing precipitation and adsorption of basic proteins. Meanwhile, its basicity helps maintain the stability of the pH gradient at the basic end of the gel strips, avoiding "compression" of the pH gradient during focusing. This enables effective focusing of basic proteins (e.g., histones, certain transcription factors) and increases their detection rate on 2-DE gels. For example, when analyzing the hepatocyte proteome, the number of protein spots in the pI 9–11 range on 2-DE gels with L-arginine added can increase from 20–30 to 50–60.

Improving Transfer and Detection in Second-Dimension SDS-PAGE

After the second-dimension electrophoresis of 2-DE, proteins need to be transferred to a blot membrane (e.g., PVDF membrane) for subsequent detection (e.g., Western blot). L-arginine can be added to the transfer buffer to reduce the binding force between proteins and the gel matrix, thereby improving transfer efficiency. Additionally, its protective effect on proteins can reduce protein denaturation during transfer, enhancing the sensitivity of Western blot detection (e.g., the signal intensity of low-abundance basic proteins can be increased by 2–5 times).

IV. Applications in Other Special Electrophoresis Scenarios

Assisting Nucleic Acid Purification in Agarose Gel Electrophoresis

When recovering DNA fragments from low-melting-point agarose gels, adding a low concentration of L-arginine (5–10 mmol/L) to the elution buffer can stabilize the DNA double-stranded structure through weak electrostatic interactions, reducing DNA degradation during elution. It also reduces the binding of DNA to residual components of the agarose gel, improving the purity of the recovered DNA (the OD260/280 ratio is closer to 1.8), which can be directly used in subsequent experiments such as PCR and enzyme digestion.

As an Auxiliary Component for pI Markers in Isoelectric Focusing (IEF)

In IEF, marker proteins with known pI values are required to calibrate the pH gradient. L-arginine can be mixed with marker proteins to reduce aggregation of the markers, ensuring the accuracy of their pI values and thus improving the precision of target protein pI determination.