9.1 TB c_1 TB Consortium - Database Group

Room 105, MBI, Los Angeles 90095

USA tbdbteam@mbi.ucla.edu TB Structural Genomics Consortium Laboratory of David Eisenberg Archiver c_2 LANL-Protein Production Facility

Los Alamos, NM 87545

USA ppteam@lanl.gov Los Alamos National Laboratory Los Alamos National Laboratory Scientist pur_1 Gel filtration 1 Gel filtration purification by FPLC from Pharmacia. It is an intermediate or final purification step to remove contaminants depending on the size difference of target and contaminants proteins. Material lists: 1) 20mM Tris-HCl buffer (pH 8.0, containing 100mM NaCl, 10mM BME)- Solution A 1M Tris-HCl, pH 8.0 (B136 Refrigerator): 80ml 4M NaCl(B136 Refrigerator): 100ml 14.3M ?-mercaptoethanol (Fisher, BP 176-100, B136 Refrigerator): 3.32ml H2O: 3816.7ml Filter with Disposable sterilization filter unit (Fisher, 09-740-3A, B136). Degas overnight. Store refrigerated. 2) 0.5M NaOH solution-Solution B Dissolve 40g NaOH (B137 Chemical shelf) in 1.8 liter D.W. Add H2O to 2000ml total volume. Filter with Disposable sterilization filter unit (Fisher, 09-740-3A, B136). Degas overnight. Store refrigerated. 3) Prepacked Superdex 75 column (Pharmacia, 17-1070-01, 2.6x 60cm, B137 Cold room) Method 1 bank 2 = Running method for protein Method 2 bank 2 = wash of column and equilibration Method 3 bank 2 = equilibration of column Procedures 1) Make sure column has been equilibrated with Solution A (use Method 3 Bank 2) - flow-rate 4ml/min - 150ml Solution A 2) Make sure that the system is connected properly. - Solution A is connected to pumpA (Pump P-500) and solution B is connected to pump B (Pump P-500). - For solvent change, SOLVENT CHANGE button on the pump can be used. To set the chart recorder (Rec 102, Pharmacia) 2-1) On the right side of the chart recorder The red pen records conductivity and the blue pen records UV absorbance. The range dial for red pen should be at 1.0V The range dial for blue pen should be at 0.1V Both CAL/VAR buttons should be in the up position. Both PEN buttons should be in the down position. Both ZERO buttons should be in the up position. Both ZERO SUPPER dials should be set to zero. 2-2) On the left side of the chart recorder The INT button should be in the up position. The MM button should be in the down position (speed setting: 1mm/min). The REC button should be in the down(on) position. The SET, HOME, GRID and both FEED buttons should all be in the up positions. 2-3) Adjust the baseline to zero while the equilibration buffer is flowing. 3) To set the fraction collector (Frac-100, Pharmacia) Press the FRACTION SIZE button, enter ~1.2(min) and press the DO/STORE. - check to make sure that 1.2min gives you a collection volume of 4.5ml (The calibration  actual collecting volume should be checked) Put 60 tubes in fraction collector and position the dispenser arm leaning on the side of the first tube. (Once the target proteinÂs peak position is identified, the collecting fraction # can be reduced depending on the previous run data) 4) To set the UV monitor (UV MII, Pharmacia) Set the AU value to 0.2 or 0.5 depending on the loaded proteinÂs concentration. 5) To run the method - Prime syringe with sterile 3X dH2O several times Suck up the over 10 ml sample and get rid of all bubbles in syringe before loading Inject 10ml sample into the injector by using a syringe and leave the syringe while sample is running. (While injecting, make sure that the measurement tube in the super loop goes up,which indicates you are loading the column!) Press the METHOD FILE and enter the bank number (#2). Press the STEP FORWARD twice and enter the method number (#1). Press DO (program will appear line by line) To view the method program, press STEP FORWARD for each line of the program. To change any line, press CHANGE or EDIT or DELETE, enter your changes, then press DO. To print a program, press STEP FORWARD until the ÂList prompt appears, then enter the method number you want and press DO. To run the method, press EXIT, then the method number you want and press DO. Ex) Bank 2 method 1 Time 0.00 conc %B 0.0 (start with solution A) 0.00 ml/min 1.5 (sets column flow rate) 0.00 cm/min 1.0 (sets chart speed in the case of external control) 0.00 port.set 6.0 (directs flow-through to waste) 0.00 valve.pos 1.1 (brings the column on line) 1.00 valve.pos 1.2 (loads sample onto column) 1.00 min/mark 5.0 (sets a mark on the chart) 51.00 port.set 6.1 (directs flow-through to fraction collector) 241.00 port.set 6.0 401.00 prt pk 1.10 After do/store button has been pushed and method is running, make an initial mark with blue ink roller to indicate injection time Write the following on the chart: - Date and time at which run was started - Absorbance (AU) setting for run - Chart speed (CS) (usually set to 2.00mm/min) - method used and bank used Check periodically during the run to make sure everything is running well (printer rolling, fractions collecting, etcÂ) Method takes about 2.5 hours to complete Enter the following information: Method and bank used: Method 1 Bank 2 AU set to: 0.5 Chart speed set at: 2mm/min Amount of protein loaded: 10/9 mlÂs 6) To wash and re-equilibrate the column after protein running, - Run the bank 2 method 2 following the procedure of step 7) Time 0.00 conc %B 100 (start with solution B) 0.00 ml/min 4.0 0.00 cm/min 0.5 0.00 port.set 6.0 0.00 valve.pos 1.2 150.00 conc % B 100 151.00 conc % B 0.0 (equilibrate the column with solution 301.0 conc % B 7) Collect fractions and load peak fractions onto a 10% Tris-glycine gel Rv2031c MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEV RAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPV GADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN protein protein domain hspX http://www.doe-mbi.ucla.edu/TB/PUBLIC/qs/qsearch.php?dowork=Rv2031c A remarkably insightful comment about this target. Mycobacterium tuberculosis TB.proteomic_information 367989 Genbank 189676547 c_2 2001-02-14 c_2 purified Los Alamos National Laboratory selected 2001-01-10 Los Alamos National Laboratory cloned 2001-02-14 Los Alamos National Laboratory expressed 2001-02-16 Los Alamos National Laboratory soluble 2001-02-24 Los Alamos National Laboratory purified 2001-03-01 MATTLPPQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEV RAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPV GADEDDIKATYDKGILTVSVAVSEGKPTEKHIQ valine 7 replaced by proline five terminal residues cleaved pur_1 purification A cat wisker was added for good luck.