Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is frequently used in protein expression analysis and other research work. It can be used to obtain electrophoretic patterns of protein samples, in combination with other Mass spectrometry-based protein identification services Analyze or purify samples for protein spectrum analysis of complex biological samples.
Biotech Park Biotechnology can provide protein separation services based on 1D SDS-PAGE or 2D SDS-PAGE according to your needs: Based on our optimized standard operating procedures, we use the Bio-Rad Mini-PROTEAN® Tetra gel system for 1D SDS-PAGE protein separation. Protein samples (5-20 µg) are diluted to a certain concentration with sample buffer and heated (boiling water bath for 5 min). According to the analysis purpose of the sample, select an appropriate concentration of gel and apply the sample to the gel wells, add electrophoresis buffer, first separate at a constant voltage of 80V for 15 min, then separate at a constant voltage of 120V for 60 min. After separation, remove the gel plate and stain the gel with Coomassie Brilliant Blue or silver stain.
2D SDS-PAGE protein separation uses IPG (pH 3-10) gel for one-dimensional isoelectric focusing, and SDS-PAGE gel for two-dimensional electrophoresis to achieve protein separation. Samples are first purified using ultrafiltration tubes, and the solution system is replaced with 2D lysis buffer (30 mM Tris-HCl, pH 8.8, containing 7 M urea, 2 M thiourea, and 4% CHAPS) for isoelectric focusing. The focused gel strips are placed face up on dry thick filter paper to squeeze out excess water, mineral oil, and sample. After swelling with buffer for 15 min, the strips are further equilibrated in equilibration buffer for 15 min. The gel strips are placed face up on the long glass plate of the gel, sealed with low-melting agarose, and transferred to the electrophoresis tank. Start with low voltage, and once the sample is completely out of the IPG strip and concentrated into a line, increase the voltage. Stop electrophoresis when the bromophenol blue indicator reaches the bottom edge. Remove the gel plate, and stain the gel with Coomassie Brilliant Blue, silver stain, or fluorescent stain. Use the Typhoon TRIO (GE Healthcare) to scan the gel image, and analyze the scanned image using Image Quant software (version 6.0, GE Healthcare) and DeCyder 5.0 (GE Healthcare).
Biotech Park 2D SDS-PAGE example
About samples:
1. Both liquid and solid samples are acceptable;
2. Generally, 2-30 µg of protein is needed for 1D SDS-PAGE, and 50-1000 µg of protein is needed for 2D SDS-PAGE;
3. Proteins stained with Coomassie Brilliant Blue, silver stain, or fluorescent stain are all acceptable.
