• 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.
One of the key factors influencing Western blot results is the extraction of proteins from cells. In practice, detergent-based buffers such as radioimmunoprecipitation assay (RIPA) buffer, along with physical disruption such as sonication, or the combination of both, have become the norm for protein extraction from cell membranes, cytoplasm, organelles, and nuclei.
IntactProtein™ Tissue and Cell Lysis Kit is formulated to solve these issues. It saves your time by avoiding the extra step of adding protease and phosphatase inhibitors; it can also preserve the structural and signal integrity of the cellular proteins. Further, this product is suitable for extracting all sizes of proteins from both adherent and suspension cells.
||1. Reagent A: 100 μl
2. Reagent B: 50 ml
||Reagent A at -20°C; Reagent B at room temperature|
"I was always having trouble dealing with extraction of large-sized proteins such as mTOR (MW 289) from cultured cells. My problem was that without sonication, the extraction efficiency of large proteins was low; with sonication, the large-sized proteins were fragmented into smaller-sized peptides. This dilemma was finally solved by using IntactProtein™ Cell-Tissue Lysis Kit. With this kit, I obtained excellent results without losing phosphorylation signals. Moreover, this kit was easy to use by simply mixing reagent A and B and lysing the cells for 15 min on ice."
HT1080 cells were grown in a 6-well plate to reach 90% confluence. Cells were washed twice with ice-cold PBS and treated as follows:
Lane 1: Reagent A + B, 30 min on ice; Lane 2: Reagent A + B, 15 min on ice; Lane 3: Reagent A + B, 5 min on ice; Lane 4: Reagent B + sonication using a VEVOR ultrasonic sonicator with 15% output power. Equal amounts of total cell lysate from each treatment was run on an SDS-PAGE gel, transferred to a PVDF membrane, and immune-stained with SMRT (160, 270 kDa) antibody. Note that 15 min on ice with the lysis kit gave the best result; whereas, Reagent B plus sonication fragmented SMRT into smaller peptides.