3-min Detergent-free Total Protein Extraction Kit (Animal Cells)

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    3-min Detergent-free Total Protein Extraction Kit (Animal Cells)

    Cat. #: P505L (50 rxn); P505 (20 rxn); P505S (5 rxn)

    Storage: Store at 4 oC Shelf Life: 12 months

    Product Description

    This kit is for rapidly extract detergent-free total proteins from cultured cells (mammalian cells, insect cells and other cultured cells) in less than 5 minute. The protein extraction buffers do not contain any detergent and EDTA. The whole procedure is less than 5 minutes.

    The extracted protein can be used for proteomics (LC/MS), IP, ELISA, 2D-gel analysis, isoelectric focusing, SDS-PAGE, immunoblottings, and other applications. This product is for research use only.

    Product Contents  

     3-min Detergent-free Total Protein Extraction Kit  (Animal Cells)

    Note

    The use of protease inhibitors is not necessary prior to extraction. However if downstream application takes significant amounts of time or the protein extract will be stored for longer period of time, addition of protease inhibitors to Buffer A is recommended. For determination of protein concentration, BCA kit (Pierce) is recommended. To study protein phosphorylation, phosphatase inhibitors (such as PhosStop from Roche) should be added to Buffer A prior to use.

    Additional Materials Required

    •  1 X PBS
    • Vortexer
    • Table-Top Microcentrifuge
    • BCA Protein Assay Kit (Pierce, Cat #. 23227) 

    Protocol: 

    A. Non-Adherent Cells

    1. Prior to protein extraction, pre-chill the buffers and the protein extraction filter cartridge with collection tube on ice.

    2. Harvest cells by low speed centrifugation. Wash the cells with 1 mL cold PBS once in a 1.5 ml microcentrifuge tube and pellet the cells by centrifugation at 3,000 rpm for 2-3 min. Aspirate the supernatant and leave a small amount of PBS (about the volume of packed cells) in the tube. Vortex the tube briefly to resuspend the cells.

    3. Add appropriate amounts of Buffer A to the cell suspension (Table 1), vortex 10 – 20 seconds to lyse the cells. Add equal volume of Buffer B to the tube and mix well by vortexing briefly. 

     Note: the presence of small amount of un-lysed cells would not affect the quality of the extraction.

    Table 1. Buffer Volume for Different Packed Cell Volumes*


    4. Transfer/pour the cell lysate to pre-chilled filter cartridge(s) in collection tube(s) and centrifuge in a microcentrifuge for 60 seconds at top speed (14,000 ~ 16,000 rpm). 

    5. Transfer the supernatant of flow through (detergent- free protein extract) to a pre-chilled microcentrifuge tube. Discard the filter cartridge. The protein is now ready for downstream applications.

    B. Adherent Cells

    1. Prior to protein extraction pre-chill the buffers and protein extraction filter cartridge (placed in collection tube) on ice.

    2. Grow adherent cells to 90-100% confluence and wash the cells once in the tissue culture plates, dishes or flasks with cold PBS, aspirate the buffer completely.

    3. Add appropriate amounts of Buffer A (Table 2), swirl to distribute the lysis buffer over the entire surface of tissue cultures, and place the tissue culture on ice for 2 min.

    Add an equal volume of Buffer B.

    Mix well by scraping the lysed cells with a pipette tip or a transfer pipette and transfer the cell lysate to pre-chilled protein extraction filter cartridge in collection tub. Centrifuge at top speed (14,000-16,000 rpm) in a microcentrifuge for 60 seconds.

    4. Immediately place the collection tube on ice. Discard the filter. The flow through cell lysate is detergent-free protein extract, and ready for downstream applications. 

    Table 2. Amount of buffers required for different amount of adherent cells 

    3-min Detergent-free Total Protein Extraction Kit  (Animal Cells)

    Remarks: This protocol is developed and validated by 101Bio’s OEM partner. Spin column based protein extraction and cell. fractionation technologies were developed by 101Bio’s OEM partner.