IntactProtein™ Cell-Tissue Lysis Kit #415
- All-in-one formula: no protease/other enzyme inhibitors or sonication required
- Ready-to-use protocol: simply mix Reagents A & B; extraction takes as little as 15 min
- Ultimate solution for large proteins: near-complete extraction of large proteins; no fragmentation due to no sonication
- Assurance and peace of mind: no loss of protein PTMs such as phosphorylation, glycosylation, ubiquitination, methylation, and acetylation
- All-around performance: suitable for mammalian cells and tissues
Reagent A (40, 100, 200 μL for #415 S, M and L)
Reagent B (20, 50, 100 mL for #415 S, M and L)
For research use only
Store Reagent A at -20°C
Store Reagent B at room temperature or 4°C (see the Technical Note below)
Background InformationOne of the key factors influencing the result of Western blotting is the quality of proteins extracted from cells and tissues. In practice, detergent-based buffers such as radioimmunoprecipitation assay (RIPA) buffer, physical disruption such as sonication, or a combination of both, have become the standard for the extraction of proteins from cell membrane, cytoplasm, organelles, and nuclei. Although RIPA buffer (with 0.1% SDS) or its substitute like NP-40 buffer (without SDS), has been widely used to lyse mammalian cells and tissues, RIPA buffer is not as effective in extracting large proteins as it is in medium and small proteins. To increase the harvest of large proteins, most laboratories combine RIPA buffer with sonication which can physically break down DNA to reduce the viscosity of the lysates. However,sonication has the potential to break down large proteins. Furthermore, to inhibit endogenous enzyme activities, inhibitors need to be added to the RIPA buffer. For example, to reduce protein degradation, protease inhibitors such as aprotinin, leupeptin, pepstatin, and PMSF need to be added to RIPA buffer immediately before use. Similarly, sodiumfluoride and sodium orthovanadate must be added to inhibit phosphatase activities.
Our IntactProtein™ Cell-Tissue Lysis Kit is formulated to solve these issues. It saves you time by avoiding adding protease, phosphatase, and other enzyme inhibitors; it can also preserve the post-translational modifications (PTMs) of the cellular proteins. Overall, this product is suitable for extracting proteins of all sizes from mammalian cells and tissues.
Experimental Protocol for Adherent Cells
- Prepare the IntactProtein™ lysis buffer by adding 2 µL of Reagent A into 1 mL of Reagent B immediately before use. Mix thoroughly by vortexing and place on ice. Tips: Calculate the volume of the lysis buffer you need as per Step 3; discard the unused buffer after use.
- Discard the cell culture medium and wash the cells twice with ice-cold PBS.
- Place the culture dish/plate on ice or ice water and add 1 mL of the premixed lysis buffer per 5x10⁶ cells (e.g. add 300 µL of lysis buffer to a 35 mm dish containing 1 x10⁶ cells). Keep the dish/plate on ice for an additional 5 min and swirl occasionally to allow the lysis buffer to completely cover the cells.
- After 5 min of lysis, scrape the cells off the dish/plate using a clean plastic scraper and collect the lysate into a centrifuge tube.
- Vortex the lysates thoroughly (3 x 10 sec) and place the lysates on ice or ice water for another 10 min to complete the lysis.
- Heat the lysates on a 95°C heat block for 5 min.
- Cool the lysates on ice or ice water for 3 min.
- Centrifuge the lysates at 13,000g for 5 min at 4°C.
- Measure the protein concentration using a NanoDrop spectrophotometer or SDS-compatible protein assay.
- Store the lysates at -20°C for future use or use immediately for further analysis. Tips: For reducing SDS-PAGE, a final concentration of 2–5% β-mercaptoethanol or 50 mM DTT, plus 0.1% bromophenol blue, must be added to the lysates. Samples should be heated at 95°C for 5 min before loading.
Experimental Protocol for Suspension Cells