Description

Optimized Protein Extraction for Western Blot Success

Achieving accurate western blot results starts with efficient protein extraction. Traditional methods like RIPA lysis buffer (with SDS) or NP-40 buffer often require supplemental sonication to extract large proteins, risking protein fragmentation. Additionally, manual addition of protease/phosphatase inhibitors adds complexity and time to workflows. The IntactProtein™ Cell-Tissue Lysis Kit revolutionizes protein preparation with a detergent-based formula designed for complete protein extraction—from small to large molecules—without compromising structural integrity.

Why Replace RIPA Buffer Western Blot Protocol? While RIPA buffer western blot workflows are common, they face limitations:

• Poor recovery of large proteins (>150 kDa) without sonication
• Risk of protein denaturation from physical disruption
• Time-consuming inhibitor supplementation

Our cell lysis kit eliminates these hurdles. Its optimized tissue lysis buffer formulation ensures near-complete extraction of proteins from adherent/suspension cells and tissues in 15 minutes—no sonication or inhibitors needed.

Key Advantages Over Traditional RIPA Lysis Buffer

• All-Size Protein Recovery: Extract large proteins (e.g., kinases, receptors) without fragmentation, outperforming standard RIPA buffer western blot prep.

• PTM Preservation: Maintain critical post-translational modifications (phosphorylation, ubiquitination) for reliable signaling analysis.

• Streamlined Workflow: Premixed Reagents A&B simplify usage—add, mix, and centrifuge. Ideal for high-throughput labs.

• Broad Compatibility: Effective for mammalian cells, frozen tissues, and challenging samples like lipid-rich membranes.

Applications

1. Western blot, IP, co-IP, and phosphoprotein studies
2. Extraction from cultured cells, xenografts, or biopsy tissues
3. Replacement for RIPA lysis buffer in SDS-PAGE and mass spec workflows

Components

1. Reagent A: 80 μl
2. Reagent B: 50 ml

Storage

Reagent A at -20°C; Reagent B at room temperature.

 

Case Study

	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 µg) 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 lntactProtein™ 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 µg) 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 µg) were immunoblotted with anti-phospho-mTOR (Ser2448) (A) and antimTOR (B) antibodies, respectively. (C) HeLa cells were lysed using the RlPA buffer or the IntactProtein™ lysis kit. Cell lysates were immunoblotted with anti-0-GlcNAG (glycosylation) antibody.