Bacterial Protein Extraction Reagent 

Bacterial cell lysis methods are generally classified into physical and chemical approaches.

Physical methods-such as sonication, high-pressure homogenization, grinding, and freeze-thaw cycles-do not introduce exogenous chemical reagents and are cost-effective with relatively high efficiency. However, they also have limitations: sonication may cause local heating and protein denaturation; grinding and freeze-thaw methods have low throughput and inconsistent lysis efficiency; and physical disruption often produces highly viscous lysates due to nucleic acid release, which may interfere with downstream protein purification.

Chemical lysis methods include organic solvents, acid-base treatment, and lysozyme-mediated digestion, and can be applied to both large- and small-volume samples with greater operational flexibility. Although acid-base lysis is rapid and efficient, the harsh conditions can disrupt native protein structures and are therefore mainly used for specialized applications such as plasmid extraction. Many chemical reagents may also inactivate proteins, limiting their applicability for functional protein studies.

MCE Bacterial Protein Extraction Reagent integrates both lysozyme and nuclease activities and is specifically formulated for E. coli lysis. It efficiently disrupts the peptidoglycan layer under mild conditions to rapidly release intracellular proteins. Simultaneously, the incorporated nucleases degrade genomic DNA/RNA, significantly reducing lysate viscosity and minimizing nucleic-acid interference, thereby preserving the native conformation and functional integrity of target proteins to the greatest extent.

This reagent is suitable for a wide range of applications, including recombinant protein expression analysis, functional protein studies, and sample preparation prior to SDS-PAGE or Western blotting. It provides an efficient and convenient solution for routine bacterial protein extraction in research laboratories.

-20°C, 2 years

The product can be stored short-term at 2-8°C for up to 2 months. For long-term storage, keep at -20°C or -80°C, and avoid repeated freeze/thaw cycles.

Harvest the bacterial cells expressing the target protein by centrifugation or other appropriate methods. Discard the culture supernatant, weigh the cell pellet, and record the wet weight.

1. Cell Resuspension

Resuspend the pellet at a ratio of 1 g wet cells to 9 mL Tris-HCl or TBS buffer (20 mM Tris, 150 mM NaCl, pH 7.6) to obtain a homogeneous bacterial suspension.

Note: PBS is not recommended, as it may mildly inhibit nuclease activity. If PBS must be used, extend the subsequent lysis time accordingly.

2. Bacterial Lysis

1) Addition of Lysis Reagent: Add the Bacterial Protein Extraction Reagent to the suspension at a ratio of 1 mL lysis reagent per 1 g wet cell mass, and mix thoroughly (select an appropriate mixing method based on total volume).

2) Lysis Conditions: Incubate the mixture at 4-25°C for 1-10 min, adjusting the temperature according to the stability of the target protein. During lysis, the suspension will gradually become clear.

Note: a. For soluble proteins, a brief lysis (~1 min) is recommended, which typically releases > 80% of the target protein into the supernatant.

b. For inclusion body proteins, extend the lysis time to ensure complete cell-wall digestion and improved inclusion body purity.

3. Post-lysis Processing

After lysis, centrifuge the sample at > 8,000 g to separate the supernatant and pellet.

For soluble proteins: collect the supernatant for downstream purification.

For inclusion body proteins: collect the pellet, perform washing and denaturation/refolding steps as needed, and proceed with an appropriate purification strategy.

1. This reagent lyses bacterial cells via lysozyme-mediated digestion of the cell wall. During this process, disaccharides derived from the peptidoglycan layer may be released and can competitively bind to the substrate-binding site of maltose-binding protein (MBP). Therefore, this product is not recommended for purification workflows involving MBP-tagged proteins.

2. The lysis process is mild and does not induce protein denaturation. Both lysozyme and nuclease included in the formulation are tag-free, ensuring that they do not interfere with downstream protein purification procedures.

3. This product does not contain EDTA, and thus will not cause nickel ion stripping during Ni-affinity chromatography.

4. Typical yields: LB cultures at laboratory scale generally produce ~3 g wet cell paste per liter. High-density fermentation can yield ~100 g wet cells per liter. If cell pellets need to be stored, keep them at -20°C or -80°C to minimize protein degradation.

5. Although enzyme activity is higher at room temperature (20-25°C), lysis is recommended at 4°C to reduce the risk of target protein degradation.

6. After lysis and centrifugation, the lysate is typically well-clarified and does not require additional depth filtration or membrane filtration. If clarification is insufficient, further clarification steps may be added as needed.

7. Ensure the bacterial pellet is fully and evenly resuspended before adding the lysis reagent, as this is critical for achieving efficient and uniform lysis.

8. This product is for R&D use only, not for drug, household, or other uses.

9. For your safety and health, please wear a lab coat and disposable gloves to operate.