IntactProtein™ Cell-Tissue Lysis Kit #415

IntactProtein™ Cell-Tissue Lysis Kit #415

$70.00Price

 

Reagents
  • Key Features

    • All-in-one formula: No protease/PTM inhibitors needed; no sonication required
    • Ready-to-use protocol: Simply mix Reagents A&B; extraction in as little as 15 min
    • Ultimate solution for large proteins: Near-complete extraction; no fragmentation
    • Assurance and peace of mind: No loss of protein PTMs such as phosphorylation
    • All-around performance: Suitable for mammalian cells and tissues
  • Kit Components

    Reagent A (40, 100, 200 μL for #415 S, M and L)

    Reagent B (20, 50, 100 mL for #415 S, M and L)

  • Application Information

    Western blotting

  • Disclaimer

    For research use only

  • Storage

    Store Reagent A at -20°C

    Store Reagent B at room temperature

  • Background Information

    Protein extraction for Western blotting is a critical step that can greatly influence the efficiency of protein detection. Commonly used detergent-based buffers such as radioimmunoprecipitation assay (RIPA) buffer or NP-40 buffers have become the norm for protein extraction from plasma membranes, organelles, cytoplasm, and nuclei. However, these buffers are not efficient at extracting large proteins, and sonication is often used with these buffers to increase the yield of large proteins. Sonication fragments the cell, which can improve protein extraction of large proteins, but oftentimes results in protein fragmentation. Additionally, RIPA and NP-40 buffers do not protect the sample from protein degradation or removal of post-translational modifications (PTM), and the procedure can give varying results from person to person.

     

    Our IntactProtein™ Cell-Tissue Lysis buffer is an innovative protein lysis and extraction buffer that is formulated to solve these issues. It is an extraction buffer with widespread applications that eliminates the uncertainty of choosing the correct buffer to efficiently extract and detect the protein of interest. It is ready-to-go, simple to use, preserves PTMs, and does not require inhibitors for proteases, phosphatases, or other PTM-degrading enzymes. Further, this product is suitable for extracting proteins of all sizes from both mammalian cells and tissues.

  • Experimental Protocols for Adherent Cells

    1. Prepare the IntactProtein™ lysis buffer by adding 2 µL of Reagent A into 1 mL of Reagent B immediately before use. Vortex the solution to mix well.
    2. Discard culture medium and wash the cells twice with ice-cold PBS.
    3. With the dish/plate on ice, add 1 mL of the mixed cell lysis buffer per 5x10⁶ cells (e.g. add 300 µL of lysis buffer to a 35 mm dish). Keep the plate/dish on ice for an additional 5 min and swirl occasionally to spread the lysis buffer.
    4. After 5 min of lysis, scrape the cells off the plate/dish and collect the lysate in a centrifuge tube.
    5. Vortex the lysates (3 x 10 sec) and place the cells on ice for an additional 10 min to complete the lysis.
    6. Heat the lysates on a 95°C heat block for 5 min.
    7. Cool the lysates on ice for 3 min.
    8. Centrifuge the lysates at 13,000g for 5 min at 4°C.
    9. Measure the protein concentration using a NanoDrop spectrophotometer or an SDS-compatible protein assay method.
    10. Store the cell lysates at -20°C or immediately use the lysates for further analysis. Note: For reducing gels, a final concentration of 2–5% β-mercaptoethanol or 50 mM DTT should be added to the lysates. The samples must be heated at 95°C for 5 min before loading.
  • Experimental Protocols for Suspended Cells

    1. Prepare the IntactProtein™ buffer by adding 2 µL of Reagent A into 1 mL of Reagent B immediately before use. Vortex the solution to mix well.
    2. Pellet the cells by centrifugation (200g for 5 min) and resuspend the cells in 10 mL of ice-cold PBS. Pellet down the cells again, discard the PBS, and resuspend the cells in the residual buffer by pipetting.
    3. Add 1 mL of cell lysis buffer per 5x10⁶ cells directly to the resuspended cells. Mix by pipetting.
    4. Follow steps 5-10 in the Experimental Protocols for Adherent Cells.
  • Experimental Protocols for Tissues

    1. In liquid nitrogen, grind tissues into fine particles with a mortar and pestle.
    2. Prepare the IntactProtein™ lysis buffer by adding 2 µL of Reagent A into 1 mL of Reagent B immediately before use. Vortex the solution to mix well.
    3. Add the frozen tissue powder into the pre-mixed IntactProtein lysis reagent at the ratio of 1g of tissue to 3 mL of lysis reagent.
    4. Homogenize the tissue according to the manufacturer’s instructions. Tip: homogenization will heat up your sample, so always keep the tubes on ice.
    5. Incubate the tissue on ice for 15 min. Tip: If you have multiple samples, keep all the samples on ice. Begin the 15 minutes after the last sample in the group is homogenized.
    6. Centrifuge at 4°C for 10 min and transfer the supernatants into clean centrifuge tubes. Supernatants contain the extracted proteins.
    7. Follow steps 6-10 in the Experimental Protocol for Adherent Cells.

Support

Orders

sales@genuinbiotech.com

Technical

info@genuinbiotech.com

2270 Kraft Drive, Suite 1000, Blacksburg, VA, 24060

Technical Support: info@genuinbiotech.com

Sales & Quotation: sales@genuinbiotech.com 

Office Hours: 8:30AM - 5:30PM EST

Phone: 540.605.9777; Fax: 540.605.9771

©2018 by GenuIN Biotech