|
|
| Line 172: |
Line 172: |
| | *Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction | | *Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction |
| | *Re-equilibrate the column with binding buffer | | *Re-equilibrate the column with binding buffer |
| - |
| |
| - | ==Bradford Protein Assay==
| |
| - |
| |
| - | ==Measuring of [http://partsregistry.org/Part:BBa_E1010 mRFP]==
| |
| - | * Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
| |
| - | * Freeze samples at -80 °C for storage
| |
| - | * To measure the samples thaw at room temperature and fill 200 µL of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)
| |
| - | * Measure the fluorescence in a platereader (we used a [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]) with following settings:
| |
| - | ** 20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm)
| |
| - | ** Measurement mode: Top
| |
| - | ** Excitation: 584 nm
| |
| - | ** Emission: 620 nm
| |
| - | ** Number of reads: 25
| |
| - | ** Manual gain: 100
| |
| - | ** Integration time: 20 µs
| |
| - |
| |
| - | ==Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)==
| |
| - |
| |
| - | This analytical Method can be used for separation and identification of proteins according to their electrophoretic mobility. The mobility is a function of length of the molecular weight. Proteins that have identical charge per unit mass due to binding of SDS results in an equal electrophoretic mobility.
| |
| - |
| |
| - | ===Pouring the polyacrylamide gel===
| |
| - |
| |
| - | *Make a master mix for the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#SDS-PAGE_gel stacking] and [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#SDS-PAGE_gel separating gel] without adding ammonium persulfate and TEMED.
| |
| - | *Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel.
| |
| - | *Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel.
| |
| - | *Layer isopropanol on top of the gel.
| |
| - | *Leave the separating gel at room temperature for >60 minutes to polymerize.
| |
| - | *Remove isopropanol and wait until the surface is dry.
| |
| - | *Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form.
| |
| - | *Insert comb without getting bubbles stuck underneath
| |
| - | *Leave the gel at room temperature for >60 minutes to polymerize.
| |
| - |
| |
| - | *For storage
| |
| - | **Remove sealing and store the gel wrapped in moistened paper towel at 4°C.
| |
| - |
| |
| - | ===Preparing the sample===
| |
| - |
| |
| - | *Mix your protein mixture 4:1 with [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#4x_Laemmli-buffer Laemmli-buffer] (30 mL protein solution + 10 mL Laemmli-buffer)
| |
| - | *Heat for 5 minutes at 95 °C.
| |
| - |
| |
| - | ===Running the gel===
| |
| - |
| |
| - | *Remove sealing, put the polymerized gel into gel box and pour [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#SDS_running_buffer SDS running buffer] into the negative and positive electrode chamber.
| |
| - | *Remove comp without destroying the gel pocket.
| |
| - | *Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample.
| |
| - | *Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel.
| |
| - | *Raise amperage up to 20 mA for running the separating gel.
| |
| - | *When the distance of the lowest molecular weight standard lane to the gel end is down to 0,5 cm stop the electrophoresis by turning off the power supply.
| |
| - |
| |
| - | ==Polyacrylamide gel staining==
| |
| - |
| |
| - | ===Colloidal Coomassie Blue staining===
| |
| - | Modified staining protocol from [http://http://newjournal.kcsnet.or.kr/main/j_search/j_abstract_view.htm?code=B021105&qpage=j_search&spage=b_bkcs&dpage=ar Kang ''et al''., 2002].
| |
| - |
| |
| - | *agitate the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Colloidal_Coomassie_Brilliant_Blue_G-250_staining_solution staining solution] at 37 °C over night to form the colloids
| |
| - | *After finishing the SDS-PAGE remove gel from gel casting form and tranfer it in to a box.
| |
| - | *Add 100 mL of the stainig solution to your polyacrylamid gel.
| |
| - | *Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation.
| |
| - | *Remove the staining solution
| |
| - | *wash the gel with dH<sub>2</sub>O
| |
| - | *Incubate the gel in ddH<sub>2</sub>O (2-6h) for bleaching the background. Shake the gel continuously during incubation. If necessary replace the colored water with new one.
| |
| - |
| |
| - | ===Silver staining===
| |
| - | Modified staining protocol from
| |
| - | *After finishing the SDS-PAGE remove gel from gel casting form and tranfer it in to a box.
| |
| - | *Add 50 mL of fixation solution and incubate the gel 1 to 24 h. The formaldehyde (37 %) must be added to the solution short time before.
| |
| - | *Remove the fixation solution and wash the gel 3 times (1-20 min) with 50 mL of 50 % (v/v) ethanol.
| |
| - | *Add 50 mL of thiosulfate solution and incubate the gel exactly for 1 min. If the gel incubates to long the background becomes to dark.
| |
| - | *Remove thiosulfate solution and wash the gel 3 times (20 s) with dH<sub>2</sub>O.
| |
| - | *Add 50 mL of impregnation solution and icubate the gel for 15 to 20 min. The formaldehyde (37 %) must be added to the solution short time before.
| |
| - | *Remove impregnation solution and wash the gel 2 times (20 s) with dH<sub>2</sub>O.
| |
| - | *Add 50 mL of developing solution and incubate the gel until protein bands become visible.
| |
| - | *Remove the developing solution immediately and wash the gel for 10 to 20 s with dH<sub>2</sub>O.
| |
| - | *Add 50 mL of stop solution and incubate the gel 10 to 20 s.
| |
| - | *Remove the stop solution and wash the gel for 1 min with dH<sub>2</sub>O.
| |
| - |
| |
| - | ==Tryptic digest of gel lanes for analysis with MALDI-TOF==
| |
| - |
| |
| - | Note:
| |
| - | *Make sure to work under a fume hood.
| |
| - | *Do not work with protective gloves to prevent contamination of your sample with platicizers.
| |
| - |
| |
| - | Reaction tubes have to be cleaned with 60% (v/v) CH<sub>3</sub>CN, 0,1% (v/v) TFA. Afterwards the solution has to be removed completely followed by evaporation of the tubes under a fume hood. Alternatively microtiter plates from Greiner® (REF 650161) can be used without washing.
| |
| - |
| |
| - | *Cut out the protein lanes of a Coomassie-stained SDS-PAGE using a clean scalpel. Gel parts are transferred to the washed reaction tubes/microtiter plate. If necessary cut the parts to smaller slices.
| |
| - | *Gel slices should be washed two times. Therefore add 200 µL 30% (v/v) acetonitrile in 0,1 M ammonium hydrogen carbonate each time and shake lightly for 10 minutes. Remove supernatant and discard to special waste.
| |
| - | *Dry gel slices at least 30 minutes in a Speedvac.
| |
| - | *Rehydrate gel slices in 15 µL Trypsin-solution followed by short centrifugation.
| |
| - | *Gel slices have to be incubated 30 minutes at room temperature, followed by incubation at 37 °C overnight.
| |
| - | *Dry gel slices at least 30 minutes in a Speedvac.
| |
| - | *According to the size of the gel slice, add 5 – 20 µL 50% (v/v) ACN / 0,1% (v/v) TFA.
| |
| - | *Samples can be used for MALDI measurement or stored at -20 °C.
| |
| - |
| |
| - | Trypsin-solution: 1 µL Trypsin + 14 µL 10 mM NH<sub>4</sub>HCO<sub>3</sub>
| |
| - | *Therefore solubilize lyophilized Trypsin from Promega® in 200 µL of provided buffer and incubate for 15 minutes at 30 °C for activation. For further use it can be stored at -20 °C.
| |
| - |
| |
| - |
| |
| - | ==Bisphenol A analysis==
| |
| - |
| |
| - | ===Extraction with ethylacetate===
| |
| - | * mix 100 µL culture supernatant with 100 µL internal standard ([[Team:Bielefeld-Germany/Protocols#Used_chemicals | bisphenol F]], 100 µg L<sup>-1</sup>)
| |
| - | * add 200 µL [[Team:Bielefeld-Germany/Protocols#Used_chemicals | ethylacetate (HPLC grade)]] for extraction
| |
| - | * vortex (30 s)
| |
| - | * centrifuge for phase separation (5 min, 5000 g)
| |
| - | * take a bit from upper phase and put it in a clean eppi
| |
| - | * SpeedVac at 40 °C to remove ethlyacetate
| |
| - | * solve remaining BPA in water (HPLC grade), vortex (30 s)
| |
| - | * solubility of BPA in water only 300 mg L<sup>-1</sup>
| |
| - | ** for LC-MS analysis of BPA, 300 mg BPA L<sup>-1</sup> is definitely enough
| |
| - | ** if you want to detect or expect higher concentrations of BPA, solve it in an acetonitrile-water-mix
| |
| - |
| |
| - | ===HPLC method===
| |
| - | * C18 reverse phase column
| |
| - | * Isocratic method: 45 % [[Team:Bielefeld-Germany/Protocols#Used_chemicals | Acetonitrile]]
| |
| - | * Flow = 0.6 mL min<sup>-1</sup>
| |
| - | * UV-detection at 227 nm
| |
| - | * Internal standard: 100 mg L<sup>-1</sup> [[Team:Bielefeld-Germany/Protocols#Used_chemicals | Bisphenol F]] (BPF)
| |
| - | * Column:
| |
| - | ** Eurospher II 100-5 C18p by [http://www.knauer.net/ Knauer]
| |
| - | ** Dimensions: 150 x 4.6 mm with precolumn
| |
| - | ** Particle size: 5 µm
| |
| - | ** Pore size: 100 Å
| |
| - | ** Material: silica gel
| |
| - | * Software:
| |
| - | ** Clarity (Version 3.0.5.505) by [http://www.dataapex.com/ Data Apex]
| |
| - | * Autosampler:
| |
| - | ** Midas by [http://www.spark.nl/ Spark Holland]
| |
| - | ** Tray cooling: 10 °C
| |
| - | * Pump:
| |
| - | ** L-6200A Intelligent Pump by [http://www.hitachi.com/ Hitachi]
| |
| - | * UV-Detector:
| |
| - | ** Series 1050 by [http://www.hp.com/ Hewlett Packard]
| |
| - |
| |
| - | [[File:Bielefeld2011 BPA + Abbauprodukt-2.JPG|900px|left|thumb|Chromatogram obtained using the above described materials and methods. Three distinctive peaks are of interest: BPA at 7,3 min retention time, BPF at 5,8 min retention time and the degradation product of BPA at 4,6 min retention time.]]
| |
| - |
| |
| - | ==NAD<sup>+</sup> detection==
| |
| - | For the [http://www.molecular-beacons.com/MB_SC_design.html design of molecular beacons] different available tools may help you so that a correct functionality is ensured under defined assay conditions. All measurements were made in the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer].
| |
| - |
| |
| - | ===Characterisation of molecular beacons===
| |
| - |
| |
| - | Preferentially, use a complementary oligonucleotide (target) as well as two enshortened oligonucleotides (split target) for [http://www.molecular-beacons.com/MB_SC_protocol.html#cap3 characterising the molecular beacon] in its closed and open state. In this way you can verify simultaneously the ineffectiveness of the split target to reach the molecular beacon`s open state.
| |
| - |
| |
| - | * Optimal wavelengths:
| |
| - | ** Determine the emission spectrum of your molecular beacon by initially using a literature value for the exctintion wavelength of the labeled fluorophore. For this add 10 µL of 1 µM molecular beacon in 200 µL [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer], mix thoroughly and wait 2 min until measuring the fluorescence with a spectrofluorometer at an defined temperature (optimally 37 °C) .
| |
| - | ** Add 10 µL of 2 µM either target or split target and shake at least 6 min until measuring the fluorescence again (when equilibrium is reached).
| |
| - | ** Repeat the procedure to determine the extinction spectrum using the self-calculated optimal emission wavelength for your fluorophore/quencher combination. For further fluorescence measurements use the extinction wavelength for which the difference between the molecular beacons's closed state and the open state signals is maximal.
| |
| - |
| |
| - | * Thermal profile analysis:
| |
| - | ** Prepare a 30 µL reaction mix composed of [https://2011.igem.org/Team:Bielefeld-Germany./Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer] and 1 µM molecular beacon. In a separate sample add 2 µM either target or split target.
| |
| - | ** Use a spectrofluorometric thermal cylcer to dertemine the fluorescence as a function of temperature. Start at 80 °C and decrease the temperature stepwise in 1 °C intervals holding each temperature for 1 min and measuring the fluorescence at the end of each step.
| |
| - |
| |
| - | * Signal-to-background ratio (S/B):
| |
| - | [[Image:Bielefeld-Germany-2011 MB S-B-ratio.png|200px|thumb|right|Signal-to-background ratio]]
| |
| - |
| |
| - | **The approach is quite similar to the one that determines the optimal wavelengths which should be used to monitor the fluorescence of the [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer] (F<sub>buffer</sub>) and the added molecular beacon (F<sub>closed</sub>) as well as the target (F<sub>open</sub>) or split target. Monitor the fluorescence at the optimal wavelengths until the equilibrium is reached each time before you add a new component.
| |
| - | ** The signal-to-background ratio can be calculated as shown on the right. Finally, compare the signal-to-background ratios after adding the target and split target to check whether the split target has an effect on the melting of the molecular beacon's hairpin structure.
| |
| - |
| |
| - | * Imaging:
| |
| - | ** Prepare a 200 µL reaction mix composed of [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer] and 500 nM molecular beacon in a PCR tube. In a separate sample add 600 µM either target or split target. Excitate the samples by a UV transilluminator and take images preferably with a camera that can detect different colours.
| |
| - |
| |
| - | ===Purification of DNA ligase===
| |
| - | The purification of the overexpressed NAD<sup>+</sup>-dependent DNA ligase gene ([https://2011.igem.org/Team:Bielefeld-Germany/ BBa_K525710]) in ''E. coli'' was performed under native conditions and Ni-NTA columns were used utilizing the recombinant protein`s C-terminal 6xHis-tag.
| |
| - |
| |
| - | *Cultivation
| |
| - | **Prepare an overnight culture of [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX] carrying the plasmid with DNA ligase ([https://2011.igem.org/Team:Bielefeld-Germany/ BBa_K525710]) in 30 ml [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#LB_medium LB medium] containing 20 μg mL<sup>-1</sup> chloramphenicol at 37 °C.
| |
| - | **Dilute the overnight culture in 100 ml [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Autoinduction_medium_for_KRX autoinduction medium] (20 μg mL<sup>-1</sup> chloramphenicol added) to an OD<sub>600</sub> = 0,1 and harvest the cells after 4 h growth at 37 °C.
| |
| - | **Use the centrifuged cells immediately for protein purification or store them at -20 °C.
| |
| - |
| |
| - | *Small-scale purification
| |
| - | **Resuspend a pellet derived from 5 ml cell culture volume in 630 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10].
| |
| - | **Add 70 μl lysozyme (10 mg/ml) as well as 25 Units benzonase nuclease and mix thoroughly.
| |
| - | **Incubate on ice for 30 min.
| |
| - | **Centrifuge the lysate for 30 min at 12000 g (4 °C). Collect the supernatant (cleared lysate).
| |
| - | **Prepare a [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 Ni-NTA spin column] ([http://www.qiagen.com/default.aspx QIAGEN]) by equilibration with 600 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10] and centrifugation for 2 min at 890 g (4 °C).
| |
| - | **Load up to 600 μl cleared lysate onto the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 Ni-NTA spin column] and centrifuge for 5 min at 270 g (4 °C). Collect the flow-through.
| |
| - | **Wash the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 Ni-NTA spin column] with 600 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-20_.28wash_buffer.29 NPI-20] and centrifuge for 2 min at 890 g (4 °C). Repeat this step and collect flow-through each time.
| |
| - | **Elute the protein with 300 μl [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-500_.28elution_buffer.29 NPI-500] and centrifuge for 2 min at 890 g (4 °C). Repeat this step and collect flow-through each time.
| |
| - | ** For centrifugal ultrafiltration use [http://www.sartorius-stedim.com/DE/en/Centrifugal-Ultrafiltration--Vivaspin-%26-Centrisart/Vivaspin-500/VS0111-VIVASPIN-500-5%2C000-MCWO-PES-25-BOX/6htcki6uex7/m8qc5rabaox/n8uj9p1cyz4/article.htm Vivaspin 500] concentrators ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) with a 5000 molecular weight cut-off PES membrane. Repeat the procedure three times by adding [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#DNA_ligase_buffer DNA ligase buffer] (4 °C) for buffer exchange (remove of imidazole).
| |
| - | ** Check each purification step and especially the purity of the protein in the final condition by [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE analysis].
| |
| - | ** Determine the protein concentration with a [https://2011.igem.org/Team:Bielefeld-Germany/ Bradford Protein Assay].
| |
| - | ** For long-time storage keep the protein at -20 °C.
| |
| - | ** If the binding conditions are not effective enough you can reduce the imidazole concentration in [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10] to 1-5 mM. If the eluate contains unspecifically bound proteins maybe a washing step with [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NPI-10_.28lysis_buffer.29 NPI-10] containing 50 mM or 100 mM imidazole could help to get higher purity.
| |
| - |
| |
| - | *Large-scale purification
| |
| - | **Resuspend a cell culture pellet in 5 ml [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer] per gramm biomass.
| |
| - | **Add 10 mM MgCl<sub>2</sub>, 1 mg/ml lysozyme] as well as 5 Units benzonase nuclease per 1 ml cell culture volume and mix thoroughly.
| |
| - | **Incubate at 4 °C for 30 min.
| |
| - | **Centrifuge the lysate for 30 min at 10000 g (4 °C). Collect the supernatant (cleared lysate).
| |
| - | **Prepare a [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&moduleid=165904 HisTrap™ FF crude column] ([http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/life-sciences_homepage GE Healthcare]) by equilibration with 5 column volumes of [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer]. The following steps have to be performed at 4 °C.
| |
| - | **Load the cleared lysate onto the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 HisTrap™ FF crude column] and collect flow-through.
| |
| - | **Wash the [http://www.qiagen.com/products/protein/purification/qiaexpressproteinpurificationsystem/ni-ntaspincolumns.aspx?ShowInfo=1 HisTrap™ FF crude column] with 10 column volumes of [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer] and collect flow-through.
| |
| - | **Elute the protein successively with 5 ml of buffers that contain different concentrations of imidazole (e.g. [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Binding_buffer binding buffer] with 40, 60, 100, 300 and 500 mM imidazole) and collect flow-through in 1 ml fractions.
| |
| - | ** For centrifugal ultrafiltration use [http://www.sartorius-stedim.com/DE/de/Zentrifugal-Ultrafiltration--Vivaspin-%26-Centrisart/Vivaspin-15/VS1501-VIVASPIN-15-10%2C000-MWCO-PES-10-BOX/6htcki6uex7/1b11mt9gskl/kn4oouwu6g3/article.htm Vivaspin 15] concentrators ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) with a 10000 molecular weight cut-off PES membrane. Repeat the procedure three times by adding [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#DNA_ligase_buffer DNA ligase buffer] (4 °C) for buffer exchange (remove of imidazole).
| |
| - | ** Check each purification step and especially the purity of the protein in the final condition by [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE analysis].
| |
| - | ** Determine the protein concentration with a [https://2011.igem.org/Team:Bielefeld-Germany/ Bradford Protein Assay].
| |
| - | ** For long-time storage keep the protein at -20 °C.
| |
| - |
| |
| - | ===Deadenylation===
| |
| - |
| |
| - | The majority of purified DNA ligase from [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX] is usually in the adenylated form. But only the apoenzyme is really useful for the [http://pubs.acs.org/doi/abs/10.1021/ac102742k NAD<sup>+</sup> bioassay] so that the AMP moiety has to be removed.
| |
| - |
| |
| - | ** Mix the purified DNA ligase with [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Deadenylation_buffer deadenylation buffer] and incubate for 10 min at 37 °C.
| |
| - | ** Use [http://www.sartorius-stedim.com/DE/de/Zentrifugal-Ultrafiltration--Vivaspin-%26-Centrisart/Vivaspin-15/VS1501-VIVASPIN-15-10%2C000-MWCO-PES-10-BOX/6htcki6uex7/1b11mt9gskl/kn4oouwu6g3/article.htm Vivaspin 15] concentrators ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) with a 10000 molecular weight cut-off PES membrane for buffer exchange at 4 °C. Repeat the procedure at least three times by adding [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#DNA_ligase_buffer DNA ligase buffer] (remove of NMN and formed NAD<sup>+</sup>).
| |
| - | ** Check the deadenylation process by [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE analysis]. If the deadenylation was successful there should not be a double band indicating both forms of DNA ligase.
| |
| - | ** Determine the protein concentration with a [https://2011.igem.org/Team:Bielefeld-Germany/ Bradford Protein Assay].
| |
| - | ** For long-time storage keep the protein at -20 °C.
| |
| - |
| |
| - | ===NAD<sup>+</sup> bioassay===
| |
| - |
| |
| - | ** Prepare a 80 μl aliquot composed of 250 nM molecular beacon and 250 nM split target in [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#NAD.2B_bioassay_buffer NAD<sup>+</sup> bioassay buffer].
| |
| - | ** Incubate for 8 min at 37 °C.
| |
| - | ** Add 6 Units of deadenylated DNA ligase (approximately ... ng).
| |
| - | ** Monitor the fluorescence with a spectrofluorometer at 37 °C and wait until it reaches equilibrium.
| |
| - | ** Add NAD<sup>+</sup> in different final concentrations (e.g. 0, 0.3, 0.5, 5, 10, 30, 40, 80, 200, 300, 500, 1000 nM), stirr shortly and monitor the fluorescence at 37 °C again.
| |
| - | ** Determine the initial enhancement rate of flourescence intensity for each NAD<sup>+</sup> concentration.
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| - | ** For the calibration curve plot the initial velocity versus the NAD<sup>+</sup> concentration.
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To obtain higher amounts and concentration of proteins we cultivated and expressed in a bioreactor. It is possible to cultivate several liters and to control temperature, pH and DO.
Modified protocol from [http://www.jbc.org/content/240/9/3685.full.pdf+html?sid=4a90c176-0ec3-489f-8c82-4734274cebf5 Neu & Heppel, 1965].
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