Protein extraction kit, contains:
20 mL 5X Aperto Cell Lysis Buffer
1.5 ml of each Solubilization Additives (5)
Lysozyme(#3L2510) and Benzonase (#3B5140) or DNase 1(#3D1401) are required for use with Aperto (not included).
Aperto is designed for use with E.coli for the lysis and extraction of proteins for analysis and further purification. It is a rapid, simple, and gentle method.
Aperto will solubilize proteins that tend to aggregate and remain in the insoluble fraction when using other commercially available products. It can be used exclusively or in conjunction with the included solubilization additives for the extraction of extremely difficult proteins. The solubilization additives stabilize proteins and prevent denaturation and subsequent aggregation and precipitation.
Brief Protocol:
For small overnight cultures:
• Induce 1-2mL of cells for protein expression.
• Harvest the cells by centrifugation at 10,000 x g in a microcentrifuge for 10 minutes and
discard the supernatant.
o Take care to remove as much supernatant as possible. Some media types can inhibit the lysozyme
used in the cell lysis buffer.
• Freeze the cell pellets for a minimum of 10 minutes at -80°C, or 20 minutes at -20°C.
• Dilute Aperto (Cat. No: 3A8600 or 3A8610) to 1X with DI water and add lysozyme (Cat. No:
3L2510) at 0.1mg/mL and Benzonase (Cat. No: 3B5140) at 25 Units/mL final concentration. Resuspsend
the cell pellet in 100 – 500µL of 1X Aperto by gentle vortexing.
• Incubate the cell suspension on a shaking platform at room temperature for 15 – 60 minutes.
• Save 10µL of the lysate and label “Total Protein Fraction.”
• Pellet the cell debris by centrifugation in a microcentrifuge at 10,000 x g for 10 minutes.
• Remove the supernatant containing soluble proteins and label as “Soluble Fraction.” Save the
cell debris pellet and label as “Insoluble Fraction.
• To run insoluble fraction, resuspend the cell debris from the previously made 1X Aperto in the
same volume as the removed supernatant.
• Add 3.3µL of an SDS PAGE Loading Buffer (Cat. No: 2S2400) to 10µL of each of the 3 samples and
heat at 95°C for 2 minutes to denature the protein.
• Load onto an SDS PAGE gel and run.
