Small Format PEP Universal Protein Purification Kit

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization. Email:marketing@medicilon.com.cn web:www.medicilon.com

Background information

Proteins play essential roles in numerous biological processes. It is always a challenge to purify a functional protein from a protein mixture because of their charge and size heterogeneity. Another challenge in protein purification is the limitation of the amount of material that can be used for the protein purification; sometimes only micrograms of proteins are available, making most of the current protein purification procedures less effective. In the PEP technology, protein mixture is first separated by a modified one-dimensional or twodimensional gel electrophoresis, this modified procedure provides good resolution while still maintaining protein function. This is followed by an efficient protein transfer step to a specially designed 384-well Protein Elution Plate (PEP). After further transfer of the samples from the PEP plate to a master 384-well microplate, functional assays can be performed using part of the sample from each well to generate an enzyme activity profile. The purity of the protein in each well can be tested by a standard SDS-PAGE (Polyacrylamide Gel Electrophoresis) after loading a portion of the sample from the master 384-well plate. If desired, Mass Spectrometry can be used to identify the protein from the well with pure protein or from the protein band on the SDS-PAGE gel. It is also possible to analyze multiple enzyme families in parallel to obtain functional profiles for each enzyme. The PEP technology can be applied to the systematic analysis of any proteins for which functional assays exist. Technology Principle One of two gel electrophoresis methods can be used for the functional protein purification depending on the complexity of the sample. If the fraction contains less than 20 proteins, it is possible to purify the protein using a modified SDS-PAGE method. On the other hand, if there are more than 20 proteins in the sample, a small format 2-D gel may be used. If there are hundreds to thousands of proteins in the mixture, a large format 2-D gel (offered as different PEP Protein Purification kit) should be used. For protein fraction with less than 20 proteins, the sample is first treated with 0.1% SDS in TrisGlycine buffer at room temperature then loaded on a SDS-PAGE gel for protein separation. For protein fraction with more than 20 protein species, 2-D gel should be used. Twodimensional (2-D) Gel Electrophoresis is a powerful technology to separate complex protein samples. In the first dimension called Isoelectric Focusing (IEF), the proteins are separated based on their isoelectric points (pI), proteins with as little as 0.02 unit pI differences could be separated, making it a high resolution method. In the second dimension, the proteins are separated based on their molecular size. Because 2-D Gel Electrophoresis is using two orthogonal parameters (charge and size) for separation and displaying the proteins in a twodimensional manner, it is one of the most powerful technologies in protein separation. In a large format gel, more than 10,000 proteins could be separated and detected with information on their relative abundance and post-translational modification acquired simultaneously. Because of these advantages, 2-D Gel Electrophoresis has been used widely in proteomics studies. However, in a typical 2-D Gel Electrophoresis, the proteins are denatured by the addition of reagents to disrupt disulfide bonds (DTT or β-mercaptoethanol), chemicals to prevent disulfide bond formation (iodoacetamide) and high concentration of SDS (typically 1%). To keep the proteins active in 2-D Gel Electrophoresis, a few modifications were made in the current PEP technology. First, no reducing reagent is used in the Isoelectric Focusing step, keeping the disulfide bonds in the proteins intact. Secondly, iodoacetamide is omitted from the process. Thirdly, much reduced SDS concentration is used in the SDS-PAGE (from 1% to 0.1%), again trying to maintain enzymatic activity. Recent studies indicated that many different enzyme families from a wide variety of organisms are active in the presence of SDS such as protein kinases, protein phosphatases, proteases, oxido-reductases, to just name a few (See References). In addition of method modification, a high resolution Protein Elution Plate (PEP) was designed. The small format PEP has 384-wells matching the current 384-well microplate dimension for ease of sample processing. For the large format PEP, the plate is composed of 4 384-well PEP thus have 1536 wells. In both the large and small format PEP, a membrane with molecular cut off of 6,000 Dalton is attached that will allow the electric current and small charged molecules to pass through but collect proteins with molecular weight large than 6,000 Dalton in the PEP wells. Furthermore, a special solution was developed for the PEP to reduce protein diffusion after the proteins are transferred from the gel to the PEP. After transfer the solutions from PEP to a deep-well master plate, the enzyme activity or protein function can be analyzed using part of the sample from the master plate and purified protein can be verified using SDS-PAGE in standard condition and identified using Mass Spectrometry.