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Overview
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Key Features
• 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
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)Please contact us at for specific academic pricing.
Background
One 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.
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- Properties
- Applications
- Reference
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Overview
Figure 1. The IntactProtein™ lysis kit demonstrates superior performance in extracting large proteins. (A)HeLa cells were lysed using the IntactProtein™ lysis kit or RIPA buffer. Total lysates (50 ug) were immunoblotted with anti-mTOR antibody. (B) HT1080 cells were lysed using the Kit, processed for the indicated period, and immunoblotted with anti-SMRT antibody. Note that sonication fragmented SMRT to smaller peptides.
Figure 2. The IntactProtein™ lysis kit is effective in extracting proteins from tissues and cells. (A) HeLa cells and mouse brains (in duplicate) were lysed using the IntactProtein™ lysis kit. Total lysates (50 ug) were immunoblotted with anti-mTOR antibody. (B) The same lysates in (A) were immunoblotted with anti-GAPDH antibody. Note that the IntactProtein™ lysis kit is suitable for extracting proteins with a broad molecular weight range from both cells and tissues.
Figure 3. The IntactProtein™ lysis kit effectively preserves protein post-translational modifications. (A-B) HeLa cells were serum starved for 16 h and stimulated with insulin (150 nM) for 5 min before protein extraction using the IntactProtein™ lysis kit. Total lysates (50 ug) were immunoblotted with anti-phospho-mTOR (Ser2448) (A) and anti- mTOR (B) antibodies, respectively. (C) HeLa cells were lysed using the RIPA buffer or the IntactProtein™ lysis kit. Cell lysates were immunoblotted with anti-O-GlcNAc (glycosylation) antibody.
Figure 4. Western blot analysis of cell lysates prepared using lysis buffers from different vendors. Protease and phosphatase inhibitor cocktail was added to Suppliers A, B, and C's lysis buffers.
Figure 5. WB analysis of tissue lysates prepared using lysis buffers from different vendors. Protease/phosphatase inhibitor cocktail was added to Suppliers A, B, and C's lysis buffers.
IntactProtein™ Cell-Tissue Lysis Kit
IntactProtein™ Cell-Tissue Lysis Kit
Catalog Number:
PPRA553804GEN
Mfr. No.:
Genu-415
Price:
$184
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Quantity:
Add to Cart:
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