For example, cellulase and pectinase for plant cells, lyticase for yeast cells, and lysozyme for bacterial cells. Often both chemical disruption and some sort of mechanical method are used together to maximize protein yields.Įnzymes that digest cell walls of plants or bacteria aid in cell lysis. For grinding with mortar and pestle, addition of splashes of liquid nitrogen will keep the sample cool. During sonication, immersion in an ice bath is sufficient. Some methods like sonication generate heat which can overheat the sample so use cooling to avoid overheating. Involve exposing cells to physical forces using dounces, sonicators, or grinding of tissue. Zwitterionic detergents like CHAPS and ionic detergents like SDS are more effective at solubilization and denature proteins during lysis. Non-ionic detergents like NP-40 are mild and nondenaturing but do not solubilize hydrophobic proteins well. Simple exposure to detergents can lyse cells that disrupt easily, like blood cells or tissue culture cells. Lastly, the appropriate choice of lysis buffer can yield higher amounts of your target protein as the detergents in the buffer can affect lysis efficiency and solubilization of different proteins.Second, consider the subcellular location of your protein of interest and choose a method that enriches for that cellular compartment.First, consider the method of cell disruption as different sample types require different methods for efficient cell lysis.Featured in well over 1500 publications and rapidly growing, Simple Western is a proven technology in all types of research applications, going far beyond what can be done with traditional Western blotting.For effective cell lysis that yields your protein of interest in a soluble form, there are a few factors to consider: More Than Just a Western Blot Alternative. With Simple Western, researchers save samples, reagents, wash buffer, time, and labor! RePlex™ on Jess and Abby lowers the cost per result compared to traditional Western blotting protocols. Researchers get accurate measurements of target proteins with Total Protein Assays and built-in Total Protein Normalization. Reproducible and Quantitative. By eliminating the variability of traditional Western blotting protocols like the gel-to-membrane transfer, Simple Western assays are highly reproducible and fully quantitative. With a processing time of 3 hours for 25 samples or 96 samples overnight, Simple Western dramatically speeds up time to results compared to traditional Western blotting protocols. High Sensitivity Multiplex. With Jess, researchers get industry-leading sensitivity in chemiluminescence and fluorescence channels and the advantage of flexible multiplex analysis.įast Time to Results. Generate up to 8 data points with just 5 µL. Low Sample Volume. Use as little as 3 µL of starting material. You can even pair your Simple Western instrument with an automated liquid handler to prepare and load samples, reagents, antibodies, and wash buffer, and set up multiple plates for analysis. Check out the Simple Western FeliX from Analytik Jena for one example on how Simple Western pairs with liquid handling automation. Just load your samples, press start, and come back to analyzed results. Resolve protein isoforms like phospho-proteins with high resolution.įully Automated. Simple Western Charge.Charge-based capillary Western blot assays use capillary isoelectric focusing (cIEF) to separate proteins by isoelectric point.Analyze small polypeptides or large target proteins like dystrophin. Simple Western Size.Size-based capillary Western blot assays use capillary electrophoresis sodium dodecyl sulfate (CE-SDS) to separate proteins by molecular weight up to 440 kDa.Unlike traditional Western blots, separated proteins are covalently attached to the capillary wall, eliminating the need to transfer proteins to a Western blotting membrane.

Traditional Western blotting steps and incubations, like sample separation, blocking, wash buffer incubations, incubations of primary and secondary antibodies, and detection of target proteins are precisely controlled inside a hands-free capillary electrophoresis instrument. Simple Western is the only fully automated Western blot processor on the market.Simple Western is a next-generation biomolecular analytical tool that seamlessly combines capillary electrophoresis with immunodetection using conventional primary antibodies and conjugated secondary antibodies.

Biorad western blot protocol