Aperto Cell Lysis and Protein Solubilization Users Manual

Transcription

1 Aperto Cell Lysis and Protein Solubilization Users Manual Revision 2 THIS MANUAL APPLIES TO THE FOLLOWING PRODUCTS: 3A8600 Aperto, 5X Cell Lysis Buffer. 20mL 3A8610 Aperto, 5X Cell Lysis Buffer. 100mL 3A8100 Aperto, 1X Cell Lysis Buffer. 100mL 3A8606 Aperto Cell Lysis and Protein Solubilization Kit

2 About the Product The Aperto Cell Lysis and Protein Solubilization System is a rapid and simple method for the lysis of E. coli and the solubilization of proteins. This product was developed in a high throughput environment and has been tested extensively vs. the 3 most popular commercial lysis buffers available. It was found that a significant number of proteins that could not be solubilized using other commercially available lysis buffers could be solubilized using Aperto. An even greater number of proteins can be solubilized when using the Solubilization Additives included in the Aperto Cell Lysis and Protein Solubilization Kit. The Aperto 5 X Cell Lysis and Protein Solubilization Kit contains 5 different Solubilization Additives that can be added to the Cell Lysis Buffer. The additives will help solubilize and stabilize your protein. About 20% of the proteins tested benefit from the addition of a Solubilization Additive. The Cell Lysis Buffer contains mild detergents and buffers that both lyse the cells and assist in the solubilization and stabilization of a broad variety of proteins. Lysis is aided by the enzyme lysozyme while the viscosity is reduced with a nuclease. About this Manual This Manual applies to the products listed below. The Aperto 5 X Cell Lysis and Protein Solublization Kit includes the 5 X Cell Lysis Buffer as well as 5 tubes of Solubilization Additives. Product Aperto, 5X Cell Lysis Buffer. 20 ml Aperto, 5X Cell Lysis Buffer. 100 ml Aperto, 1X Cell Lysis Buffer. 100 ml Aperto 5 X Cell Lysis and Protein Solubilization Kit containing 50 ml of 5X solution and 1.5ml each of the Solubility Additives SA 1, SA 2, SA 3, SA 4, SA 5 Part Number 3A8600 3A8610 3A8100 3A8606 For a complete list of products referenced in this manual and related products that will assist you in cell growth, gene expression and protein purification please visit the following link: If you are already familiar with Aperto then the Brief Protocol in the following section should be sufficient, otherwise the Detailed Protocol should be followed. Please take a few minutes and read the General Considerations and Growth Conditions sections before you begin. They can save you a lot of head scratching.

3 Brief Protocol Below is a brief protocol for small overnight cultures. Please read the General Considerations (Appendix 1) and the Detailed Protocol sections before using this product to ensure success. 1) Induce 1 2 ml of cells for protein expression. 2) Harvest the cells by centrifugation at 10,000 x g in a microcentrifuge for 10 min and discard the supernatant. Note: Take care to remove as much supernatant as possible. Some media types can inhibit the lysozyme used in the cell lysis buffer 3) Freeze the cell pellets for a minimum of 10 min at 80 C or 20 min at 20 C. 4) Dilute Aperto to 1X with DI water and add lysozyme (Teknova Cat. No: 3L2510) at 0.1 mg/ml and Benzonase (Teknova Cat. No: 3B5140) at 25 Units/ml final concentration. Resuspend the cell pellet in µl of 1X Aperto by gentle vortexing. 5) Incubate the cell suspension on a shaking platform at room temperature for min. 6) Save 10 µl of the lysate and label as Total Protein Fraction. 7) Pellet the cell debris by centrifugation in a microcentrifuge at 10,000 x g for 10 min. 8) Remove the supernatant containing soluble proteins and label as Soluble Fraction. Save the cell debris pellet and label as Insoluble Fraction 9) To run insoluble fraction, resuspend the cell debris from the previously made 1X Aperto in the same volume as the removed supernatant. 10) Add 3.3 µl of SDS PAGE Loading Buffer to 10 µl of each of the 3 samples and heat at 95 0 C for 2 min to denature the protein. 11) Load onto and SDS PAGE gel and run

4 Detailed Protocol The following protocol is for screening small cultures. 1. Induce 1 2ml cells for protein expression 2. Harvest cells by centrifugation at ~10,000 x g for 10 min. 3. Remove media completely. Some media formulations will inhibit lysozyme if too much is left behind. Azure and Studier 2P media generally will not. 4. Freeze Cell Pellets. Pellets that have been previously frozen lyse better than those that are used fresh. 10 min at 80 C is sufficient. 5. Preparing Aperto 1X Protein Extraction Reagent. Aperto is supplied as 1X and 5X solutions. The 5X solution is provided in the kit so you may include protease inhibitors, reducing agents, and Solubilization Additives in the lysis buffer. To use the Solubilization Additives you will need to use the 5X solution. The example below illustrates a typical 100 µl experiment where SA 2 (Solubilization Additive 2), DTT, and PMSF are added to the lysis solution. Ingredient DI water Final Make up to the required volume (100 μl in this case) Per 1 2ml cell pellet example 24 µl Aperto 5X 5X 20 µl Lysozyme 10 mg/ml (Teknova Cat. No: 3L2510) DNaseI 2000 Units/ml or Benzonase 0.1 mg / ml 1 µl 100 Units / ml (DNase1) or 25 Units/ ml Benzonase 5 µl (DNase1) ORDER OF ADDITION Solubilization Additive 2 (SA 2) 50% 50 µl 1 M DTT 20 mm 2 µl 1 M PMSF 20 mm 2 µl

5 Five Solubilization Additives are included in your kit. They should be used when your protein of interest is not visible by SDS PAGE or the enzymatic activity of your protein is not optimal. When making up 1X Aperto Lysis Mix you may replace up to 50% of the solution with a Solubilization Additive. The useful range of addition for the Solubilization Additives is 10 50% of the final volume. Solubilization Additives Useful Range Recommended starting concentration SA % 50% SA % 50% SA % 50% SA % 50% SA % 50% 6. Gently vortex the cells to resuspend them. Lysozyme, Benzonase, and DNase I are enzymes and as such can be damaged by excessive vortexing. Benzonase and DNase I can be used interchangeably here. Their purpose is to degrade DNA and reduce viscosity in the sample for ease of handling and both work equally well. 7. Incubate the cell suspension 15 min at room temperature, with gentle rocking or rotating. Note: longer incubation times (30 to 60 minutes) will aid in solubilizing proteins that otherwise would be difficult to recover in the soluble fraction. For difficult proteins it is useful to take samples of the total and soluble protein fraction throughout the extended incubation period and analyze them by SDS PAGE. You will see proteins becoming soluble and moving into the soluble fraction. 8. Optional: collect a small sample of the complete lysis mix for the Total fraction to analyze by SDS PAGE. 9. Centrifuge lysis mixture for 10 min at 10,000 x g to remove cell debris. 10. Transfer supernatant to a fresh tube. 11. Optional: Collect a small sample of the supernatant for the soluble fraction to analyze by SDS PAGE. 12. Optional: Re suspend the pellet from step 10 in Aperto (same volume as the removed supernatant) and collect a small sample for the insoluble fraction to analyze by SDS PAGE. The clarified protein extract is now ready for further purifications, assays, or any other desired use.

6 General Considerations for Extracting and Solubilizing Proteins The Effect of Growth Conditions. If your cloned/expressed protein has an unknown effect on its E.coli host then it is advisable to initially grow the cells under non inducing conditions. When using the lac/t7 system we recommend using Studier ZYM 5052 AutoInduction Media (Teknova Cat. No: 3S2000). To promote proper folding E.coli is grown at reduced temperatures (18 30 C). Growth conditions that we employ when expressing uncharacterized proteins for the first time are given below. Lysis Incubation Times. To solubilize hard to purify proteins, somewhat longer incubations during the lysis step is often helpful. Though 15 min is sufficient in general, an incubation time of min will liberate more proteins from the cell debris. If you over lyse the cells however the lanes on SDS PAGE will begin to look smeared. Solubilization Additives. Five Solubilization Additives are included in this kit. If your protein of interest doesn t appear in sufficient concentrations when using the standard lysis buffer (or fails to appear at all!!!) do not fret as it is probably still there. Repeat the lysis with the addition of the additives. Set up 6 separate lysis reactions, one as a control and the other 5 to test each of the Solubilization Additives. Add equal volumes of Solubilization Additive to Aperto (50% final). Growth Conditions The following conditions are the recommended growth conditions for screening small volume cultures in deep well plates. When working with a new protein we typically evaluate a number of lysis conditions using the various solubilization additives that are supplied in the Aperto 5X From a Frozen Glycerol Stock 1) Prepare 500 µl of Azure media supplemented with 1% glucose and antibiotic per well in a 2ml 96 well deep well plate (DWP). Inoculate with 10 µl of a glycerol cell stock. This is the first o/n (overnight culture). 2) Grow for about 16 hrs at 37 C. 3) Transfer 25 µl of this culture to a fresh 96 well plate containing 500 µl Azure media with 1% glucose and antibiotics per well. This is the second o/n. 4) Grow for 16 hrs at C, then raise temp to 37 0 C and continue to grow for 3 hrs only (37 0 C increases cell mass. This is still. non inducing conditions) 5) Transfer 25 µl of the second o/n culture to a fresh 96 well plate containing Studier 2P Autoinduction media + antibiotic and grow/induce at C for hrs (500 ul/well). 6) Measure and record OD600. While the OD600 varies significantly from clone to clone typical values range from ) Spin down DWP at 4000 x g for 15 min and freeze for a minimum of 10 min at 80 0 C or 20 min at 20 0 C. 8) Lyse cells using Aperto Cell Lysis Protocol.

7 From a Single Colony on a Plate 1) Aliquot 500 µl of Azure media supplemented with 1% glucose and antibiotic per well in a 2 ml deep well 96 well plate. Inoculate with a single colony from a fresh plate. 2) Grow for 16 hrs at C, then raise temp to 37 0 C and continue to grow for 3 hrs only. (37 0 C increases cell mass. This is still non inducing conditions) 3) Transfer 25 µl of this culture to a fresh 96 well plate containing Studier 2P Autoinduction media + antibiotic and grow at C for hrs (500 µl/well). 4) Harvest cells by centrifugation at 4000 x g for 15 minutes and freeze for a minimum of 10 min at 80 0 C or 20 min at 20 0 C. 5) Lyse cells using Aperto Cell Lysis Protocol. References 1. F.W. Studier (2005) Protein production by auto-induction in high-density shaking cultures. Prot. Exp. Pur. 41, Shokri A1, Sandén AM, Larsson G.Growth rate dependent changes in Escherichia coli membrane structure and protein leakage.applmicrobiolbiotechnol. 58(3),386 92, (2002) 3. Atefeh Shokri1 and Gen Larsson. Characterisation of the Escherichia coli membrane structure and function during fedbatchcultivation.microb Cell Fact. 3: 9, (2004)