Jan 25, 05

His Bind Kit (Novagen)

(1) Prepare 5ml of 1X Charge buffer (stock is 8X= 400mM NiSO4): 0.625ml of the stock + 4.375ml DH2O. (2) Prepare 13ml of 1X Binding buffer (stock is 8X = 40mM imidazole, 4M NaCl, 160mM Tris-HCl, pH 7.9): 1.625ml of the stock + 11.375ml DH2O (3) Prepare 6ml of 1X Washing buffer (Stock is 8X= 480mM imidazole, 4M NaCl, 160mM Tris-HCl, pH 7.9): 0.75ml of the stock + 5.25ml DH2O (4) Prepare 6ml of 1X Elute buffer (Stock is 4X = 4M imidazole, 2M NaCl, 80mM TrisHCl, pH 7.9) 1.5ml of the stock + 4.5ml DH2O (5) Prepare Strip buffer (Stock is 4X = 400mM EDTA, 2M NaCl, 80mM Tris-HCl, pH 7.9) Working buffers (under native conditions): * 1X Binding Buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl) * Washing Buffer (60mM imidazole, 0.5M NaCl, 20mM Tris-HCl) * Elute Buffer (1M Imidazole, 0.5M NaCl, 20mM Tris-HCl). Working buffers (under denaturating conditions): * 1X Binding Buffer (5mM imidazole, 6M Urea (4.68g for 13ml),0.5M NaCl, 20mM Tris-HCl) * Washing Buffer (20mM imidazole, 6M Urea (2.16g for 6ml), 0.5M NaCl, 20mM Tris-HCl), since target proteins tend to elute at lower [imidazole] in the presence of 6M Urea or Guanidine HCl. * Elute Buffer (1M Imidazole, 6M Urea (2.16g for 6ml), 0.5M NaCl, 20mM TrisHCl). ------------------------------------------------* To prepare stock 100ml of local 8X binding buffer (40mM Imidazole, 4M NaCl, 160mM Tris-Cl, pH 7.9): 0.272g Imidazole (MWt: 68.08g/mol), 23.37 NaCl (58.45g/mol) and 1.93g Tris (121.14g/mol) in 100ml and adjust the pH to 7.9. * To prepare 100ml 1X binding buffer from 8X binding buffer: Take 12.5ml of 8X buffer +87.5ml H2O. * To prepare 6M Urea containing 1XBinding buffer: Take 12.5ml of 8X buffer + 36.036g Urea (MWt: 60.06g/mol) and complete the volume to 100ml with H2O. This will give 5mM Imidazole, 0.5M NaCl, 20mM Tris and 6M Urea. * Denaturing 1X Washing buffer contains (6M Urea, 20mM imidazole (rather than 60mM), 20mM Tris and 0.5M NaCl) : 7.2g urea, 0.5845g NaCl, 0.0272g Imidazole and 0.04845g Tris in 20ml water. Adjust pH to 7.9 * Denaturing 1X Elution buffer (1M Imidazole, 6M Urea, 0.5M NaCl, 20mM Tris-HCl). 7.2g urea, 0.5845g NaCl, 1.36g Imidazole and 0.04845g Tris in 20ml water. Adjust pH to 7.9 Procedure: Gently mix the His Bind Resin until completely suspended. The Resin bottle is 50% slurry (means if you want to pack a 2ml column, take 4ml of the well-mixed resin). Allow the resin to settle under gravity force.

When the level of storage buffer drops to the top of the column, add the following (in order): a- 3ml of DH2O b- 5ml of 1X Charge buffer c- 3ml of 1X Binding buffer When the Binding buffer is lower the top of the column, add the prepared extract (lysate soluble fraction). The flow rate should be 10 times the length of the column per hour for efficient purification (i.e. for 2ml packed column, allow 20ml/hour). Wash the column with 10ml of 1X Binding buffer. Wash the column with 6ml of 1X Washing buffer. Elute with 6ml of 1X Elute buffer. Inclusion body purification The procedure isolates and washes inclusion bodies from E. coli in 1XBinding Buffer (diluted from the 8X supplied stock or prepare according to buffer compositions given on page 4) to remove contaminating proteins followed by suspension in 1X Binding Buffer plus either 6 M guanidine-HCl or 6 M urea to solubilize the protein. 1. Harvest the cells by centrifugation at 10,000 g for 10 min. Decant the supernatant and allow the cell pellet to drain as completely as possible. Resuspend the cells in 40 ml 1X Binding Buffer per 100 ml culture volume (without denaturant). 2. Sonicate briefly as described above to resuspend the pellet thoroughly and to shear the DNA. 3. Centrifuge at 5,000 g for 15 min to collect the inclusion bodies and cellular debris while leaving other proteins in solution. 4. Remove the supernatant and resuspend the pellet in 20 ml 1X Binding Buffer per 100 ml culture volume (without denaturant). Repeat step 3. Sonication may be necessary to resuspend the pellet. Sometimes repeating this step several times releases more trapped proteins. 5. Remove the supernatant from the final centrifugation and resuspend the pellet in 5 ml 1X Binding Buffer containing either 6 M guanidine-HCl or 6 M urea per 100 ml culture volume. See page 10 for directions on making the 1X denaturing Bind Buffer. 6. Incubate on ice for 1 h to completely dissolve the protein. Remove insoluble material by centrifugation at 16,000 g for 30 min. Filter the supernatant through a 0.45 micron membrane prior to performing HisBind purification. Note: rLysozyme Solutionmay be added (although it is not required) at step 4 for processing insoluble protein fractions. Lysozyme has been shown to improve the purity of inclusion body preparations by digesting cell wall debris.

Add rLysozyme to the resuspended material in 1X Binding Buffer using a final concentration of 1 KU/ml. Vortex gently to mix, incubate for 510 min and then proceed with centrifugation. Purification under denaturing conditions If the target protein is found in the inclusion body fraction, HisBind Resin purification can be performed under denaturing conditions at room temperature. 1. The inclusion body fraction is solubilized in 1X Binding Buffer including a denaturant (6 M guanidine-HCl or 6 M urea) according to protocols in the Cell Extract Preparation section. 2. The HisBind Resin is charged and equilibrated as described previously using 1X Binding Buffer with a denaturant. The Charge Buffer should not contain a denaturant. 3. Purification under denaturing conditions is identical to the above procedure with the modification that Wash and Elution Buffers should contain a denaturant. A lower imidazole concentration (20 mM) should be used in the wash buffer since target proteins tend to elute at lower imidazole concentrations in the presence of 6 M guanidine-HCl or 6 M urea. Buffer preparation for denaturing purification To prepare buffers for denaturing purification, add solid guanidine-HCl or urea directly to an aliquot of concentrated buffers, bring up to 90% of the final volume with deionized water, and stir until the solid is dissolved. Adjust the pH to 7.9 with either HCl or NaOH and bring to the final volume with deionized water. For example, to make 100 ml of 1X Binding Buffer with 6M urea combine 12.5 ml 8X Binding Buffer and 36 g of urea and bring up to 90 ml with deionized water. Once the urea is dissolved adjust the pH to 7.9 and bring to 100 ml with deionized water. To make the 20 mM imidazole Wash Buffer, combine 11 ml of 1X Binding Buffer with 4.1 ml of 1X Wash Buffer, both including denaturant. Urea solutions must be made fresh and used promptly because urea decomposes to form cyanate ions, which can covalently modify primary amines on the target protein. When urea is used, samples may be mixed with sample buffer and loaded directly on an SDS polyacrylamide gel, whereas samples in 6M guanidine must be diluted 1:5 in water or dialyzed before running on an SDS polyacrylamide gel. Resin regeneration When elution is complete, the HisBind Resin can be regenerated for reuse. This process can be carried out many times. However, because some small amounts of protein may not be released with EDTA treatment, it is advisable to use a different sample of resin for each different protein studied. In step 13, it is important to use 3 volumes of water to completely remove the EDTA. Following the last elution step, the column should be washed with 3 bed volumes of 1X Strip Buffer. The presence of 100 mM EDTA in the Strip Buffer will prevent bacterial growth. The column should be stored in this solution and recharged as in Resin preparation before use.

When the flow rate of a column slows noticeably or the resin does not turn a strong blue-green color when Charge Buffer is added, then it is time to clean the resin more thoroughly. One volume is equivalent to the settled bed volume. (1) 2 vol 6 M guanidine-HCl, 0.2 M acetic acid (2) 2 vol water (3) 1 vol 2% SDS (4) 1 vol 25% ethanol (5) 1 vol 50% ethanol (6) 1 vol 75% ethanol (7) 5 vol 100% ethanol (8) 1 vol 75% ethanol (9) 1 vol 50% ethanol (10) 1 vol 25% ethanol (11) 1 vol water (12) 5 vol 100 mM EDTA, pH 8.0 (13) 3 vol water (14) 3 vol 20% ethanol. Store at 4C.

To prepare reagents for cloum generation:

(1) 0.2M acetic acid (10ml): pipet 120l of stock conc acetic acid (60.05g/mol) in 10ml distilled water. (2) 2% SDS from 10% stock: take 2ml of 10% stock + 8ml water. (3) 0.1M sodium-EDTA, pH 8.0: add 0.372g of EDTA (372.2g/mol) in 10ml water. Dissolve well. Adjust the pH to 8.0 Other ethanol solutions (25, 50 and 75%) are prepared from the absolute ethanol stock.

His column to trap His.S.Tag protein in pET30a alone (without an insert)

To clear antibodies which are S.Tag specific in an attempt to get more specific antibody population to SmGPCR, we first used Rosetta/pET30a alone glycerol stock to amplify it, harvest the induced sequence (with no il3 insert) and purify the His/S. tags using His.kit from Novagen. We will use the purified His/S.Tag sequence to treat the SmGPCR IgG to preclear any S.tag antibodies. Procedure: (1) On Sunday, 300l of Rosetta/pET30a glycerol stock was added to 100ml of LB/Kan/Chloroamph medium in 250ml flask and shaked O/N at 250rpm/37C. (2) On Monday, 3 new glycerol stocks were made from overnight culture (with 50l glycerol + 450l culture), vortexed and kept at -121C.

(3) The rest of overnight culture was poured in 1L LB/Kan/Chloramph and shaked. After 2hrs, the reading of spectrophometer was 0.55 (blanked to LB/Kan/Chloroamph alone). (4) After 3-3.5hrs, the reading was 0.98 and IPTG was added to induce the Rosetta cells at a final conc of 0.7mM. The culture was kept for another 3hrs after induction. (optional) before harvesting, you can keep some induced culture in a small tube. This can serve you to determine where your target peptide is present (i.e. in the medium as secretory product, associated with the membrane or in the cytoplasm). (5) To harvest, pour the induced culture in centrifuge 200ml tubes, balance them and spin at 10,000 xg for 10min/4C. Keep the pellet. (6) Resuspend the pellet in ice-cold 50mM Tris buffer, pH 8.0 (7) Centrifuge and keep the pellet. Store at -121C (or -80C) till future processing.

Bacterial lysis to extract the proteins:

(1) On Tuesday, The pellet is thawed and resuspended in ice-cold 1X bind buffer. (2) Spin at 10,000xg for 10min/4C. (3) Add 30ml of 1X Binding buffer + protease inhibitor, sonicate for 15min to shear DNA. (4) Spin at 10,000xg for 10min/4C to collect the inclusion bodies. (5) Collect the supernatant and keep it in the freezer (just to look later if the S.tagged peptide) is in the soluble fraction. (6) Add 30ml of 6M urea-bind buffer to the pellet and resuspend it. Incubate on ice for 1hr with occasional shaking. (7) After incubation, spin at 16,000xg for 25min/4C and use the supernatant (which contains the urea denatured, soluble inclusion bodies in the His. column. Keep the pellet in the freezer. _______________________________________________