1D SDS-PAGE, IEF Service

Characterize and isolate proteins analytically is crucial for studying protein's function. Creative Proteomics is more comprehensive and advanced in provide protein gel services, including SDS-PAGE protein identification, IEF, and native PAGE analysis. The gel electrophoresis technique is a simple, rapid, cost-effective, and highly sensitive method that holds significant importance in the field of protein studies.

What is 1D SDS-PAGE?

The technique of SDS-PAGE (Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis) is widely utilized in the field of protein research for the purpose of protein separation and facilitating scientific visualization. Proteins separate in gel is primarily achieved by leveraging the molecular sieve effect of polyacrylamide. Proteins migration rate in the electrical field mainly depend on field strength, solubility, net charge, molecular weight and shape of proteins, ionic strength, and pore size and rigidity of SDS-PAGE. The following is a more detailed explanation of the working principle of SDS-PAGE: How Does SDS-PAGE Work?. Briefly, by combining acrylamide and bisacrylamide in a specific ratio, along with the addition of 0.1% SDS, APS (ammonium persulfate), and TEMED (N, N, N', N'-tetramethylenediamine), a crosslinked polymer network gel is formed. The concentration or percentage of polyacrylamide affects the pore size and rigidity of SDS-PAGE. Low-percentage gels are better for resolving large proteins, and high-percentage gels are better for resolving small proteins. After adding protein sample loading buffer to the samples and boiling them for 5-10 minutes, the proteins are denatured, resulting in linearization of their structure and acquisition of a complete negative charge due to the presence of sodium dodecyl sulfate (SDS) in the buffer. Proteins complete denaturation requires the buffer contains of β-mercaptoethanol (β-ME) or dithiothreitol (DTT) to disrupt disulfide bonds. Thus, when a current is applied, negatively charged SDS-coated proteins move from the negative cathode towards the positive anode. For protein visualization and better conduct downstream experiments, the Coomassie staining (G250 or R250) or silver staining methods or western blot are commonly employed.

Figure 1. Protein separation using SDS-PAGE technique.

What is IEF?

The technique of isoelectric focusing (IEF) is a mature modern biochemical experimental technology used for the separation of proteins based on their isoelectric point (pI). Currently, isoelectric focusing technology has the capability to differentiate biomolecules with a minimal difference of only 0.001 pH units in their pI. The high resolution, excellent repeatability, large sample capacity, and simple and rapid operation of this technique have made it extensively utilized in the fields of biochemistry, molecular biology, and clinical medical research. IEF utilizes the disparity in proteins or peptides' pI to effectively separate and detect them through a stable, continuous, and linear pH gradient established within polyacrylamide or agarose gel. Since proteins are amphoteric compounds, the charge they carry is related to the pH value of the medium. The protein with a net charge migrates in the opposite polarity direction during electrophoresis (pH>pI, protein releases protons and is negatively charged and moves towards the anode; pH=pI, protein is uncharged and ceases movement; pH<pI, protein is positively charged and migrates towards the cathode). When the protein reaches its pI position in the gel, the current reaches its minimum and the protein ceases to move.

According to the different pH gradient establishment methods, it is divided into two types: CAPG-IEF (carrier ampholytes pH gradient) and IPG-IEF (immobilized pH gradient). When a carrier ampholyte component is added to a certain medium, a pH gradient gradually forms from the anode to the cathode upon application of voltage. This is CAPG-IEF. The IPG-IEF using an immobilized pH gradient gel, which avoids problems such as long focusing time, unstable pH gradient, and cathode drift caused by carrier ampholytes. In comparison, reports showed that the resolution of IPG-IEF is better than CAPG-IEF.

Figure 2. Protein separation using IEF method.

The applications of 1D SDS-PAGE

1. Estimate the molecular weight of unknown proteins.
2. Monitor protein purifications, check the purity of samples.
3. Proteins relative quantification.
4. Quality control and test proteins sample abundance.
5. Disulfide bonds identification and PTMs.
6. Detect or confirm the interactions between proteins.

The applications of IEF

1. Detect or verify proteins pI.
2. Monitor microheterogeneity and purity in purified protein.
3. Test proteins isoforms.
4. Combinate IEF with 1 D SDS-PAGE enables high-resolution 2D PAGE electrophoresis.

How to place an order

Creative Proteomics possesses the professional expertise and proficiency to provide a customized experimental scheme that aligns precisely with your specific requirements and projects. Experienced technical team with strict and skillful techniques, our service is high-quality for reliable results. Please feel free to contact us via email (info@creative-proteomics.com) for a comprehensive discussion regarding your specific requirements. Our customer service representatives are available round the clock, seven days a week.

Proteomic Approaches for Studying Alcoholism and Alcohol-Induced Organ Damage

Journal: Alcohol Research & Health

Published: 2008

Main Technology: Shotgun Proteomics, two-dimensional gel electrophoresis (2-DE)

Figure 1. The principle of two-dimensional gel electrophoresis. Protein extracts obtained from cells or tissues first are loaded on a thin gel strip and under the influence of an electric current separated according to their electric charge (isoelectric point). The gel strip then is loaded onto another gel and exposed to a second electric current flowing in a direction perpendicular to the first one. Under these conditions, the proteins migrate from the initial gel strip into the second gel, with the distance traveled depending on the mass of the proteins. With this approach, the potentially thousands of different proteins in an extract can be separated into individual spots that can then be visualized and cut from the gel so that the proteins can be extracted for further analysis.

1. How do we determine whether a large protein is a long single peptide chain or a polypeptide chain composed of the same or different peptides?

We can employ both reductive and non-reductive approaches for protein sample preparation prior to loading onto SDS-PAGE. The reductive method involves the addition of a protein sample loading buffer containing DTT or β-ME, which serves to disrupt any pre-existing disulfide bonds within the peptide chains. Conversely, the non-reductive method employs a buffer without any reducing agents.

2. How can we clearly distinguish both large proteins and small proteins in the same gel?

Loading your proteins samples into a gradient SDS-PAGE (from low to high percentage of polyacrylamide) is preferable. However, preparing a gradient SDS-PAGE requires specific apparatus. Simple methods to meet simple demands. For example, if you need to separate the 120 and 100 kDa proteins, as well as the 20 and 25 kDa proteins on the same gel, we can create a resolving gel with two parts: the bottom part would be 15%, while the upper part would be 8%.

3. Which method should be selected for visualizing proteins in gel, Coomassie staining or silver staining?

Both methods are compatible for mass spectrum analysis. However, in terms of sensitivity, the silver staining method is generally considered to be more sensitive, reaching 0.1 ng, while the staining is generally considered to be able to stain only a minimum of 50-100 ng of protein bands.

4. What is the 2D PAGE Electrophoresis?

The techniques combination of IEF and 1D SDS-PAGE constitutes 2D PAGE electrophoresis. For more detailed information please see 2D Electrophoresis Service.