Team:Bielefeld-Germany/Protocols/Analytics

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Revision as of 18:28, 21 September 2011

Analytics: This is a list of our used analytical methods.

Bradford Protein Assay

For determination of protein concentrations in protein extracts from bacterial cell lysates or after protein purification steps a Bradford Protein Assay can be applied. For each sample three replicates and at least two dilutions should be measured.

• Prepare differentially concentrated BSA solutions in a range of 0-25 µg/mL for a BSA calibration curve.
• Fill a 96-well plate with 160 µL of each sample.
• Add 40 µL of Bradford [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=500-0205 Protein Assay Reagent] to each well and mix thoroughly by stirring as well as pipetting up and down.
• Incubate for 10 min at room temperature.
• Use a plate reader for the measurement of absorbance at 595 nm.
• Calculate the protein concentration with the help of the BSA calibration curve.

Fluorescence measurements

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 biological samples at -80 °C for storage, keep cell-free at 4 °C in the dark
• 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

Measuring of [http://partsregistry.org/Part:BBa_J18931 mCitrine]

• Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
• Freeze biological samples at -80 °C for storage, keep cell-free at 4 °C in the dark
• 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: 488 nm
  • Emission: 529 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 stacking and 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 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 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 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 dH₂O
• Incubate the gel in ddH₂O (2-6 h) for bleaching the background. Shake the gel continuously during incubation. If necessary replace the colored water with new one.

Silver staining

• After finishing the SDS-PAGE remove gel from gel casting form and transfer 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 dH₂O.
• 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 dH₂O.
• Add 50 mL of fixation 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 dH₂O.
• 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 dH₂O.

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) CH₃CN, 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 NH₄HCO₃

• 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 ( bisphenol F, 100 µg L^-1)
• add 200 µL 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^-1
  • for LC-MS analysis of BPA, 300 mg BPA L^-1 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 % Acetonitrile
• Flow = 0.6 mL min^-1
• UV-detection at 227 nm
• Internal standard: 100 mg L^-1 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]

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.

LC-ESI-qTOF-MS(-MS)

HPLC method

• Column: C18 reverse phase column (Knauer [http://beta.knauer.net/products/column-detail-view/productdetail/vertex_plus_column_50_x_2_mm_blueorchid_175_18_c18-1.html Blue Orchid])
  • dimension: 50 x 2 mm
  • Pore size: 175 Å
  • Particle size: 1.8 µm
• Flow: 0.4 mL min^-1
• Column temperature: 30 °C
• Gradient:
  • 0 - 1.05 min: 45 % acetonitrile
  • 2.55 min: 95 % acetonitrile
  • 6.00 min: 95 % acetonitrile
  • 6.15 min: 45 % acetonitrile
  • 12.00 min: 45 % acetonitrile
• VWR Hitachi LaChrom ULTRA HPLC equipment
• Software: HyStar 3.2, HyStarPP, mircrOTOF Control

Ionization method

• Using Bruker Daltonics micrOTOF_Q
• ESI in negative mode
• Mass range: 50 - 1500 m/z
• End plate offset: - 500 V, 107 nA
• Capillary: 2500 V, 4 nA
• Nebulizer: 3 bar
• Dry gas: 8 L min^-1
• Quadrupole
  • Ion energy: 5 eV
  • Low mass: 100 m/z
• Collision energy: 10 eV
• Collision RF: 150 Vpp
• Transfer time: 70 µs
• Pre puls storage: 7 µs

MS-MS

• Isolated mass: 243.1 +/- 0.1
• Collision energy: 30 eV

NAD⁺ detection

Characterization of molecular beacons

Molecular beacons are nucleic acid probes that fluoresce upon hybridization to a complementary target. To check whether they are suitable for a NAD⁺ bioassay involving NAD⁺-dependent DNA ligase from Escherichia coli (LigA) they can be characterized by thermal profile analysis and determination of the signal-to-background ratio. Detailed background information, tools for the correct design of molecular beacons, a list of synthesis companies as well as protocols for the synthesis and characterization of molecular beacons can be found [http://www.molecular-beacons.com/default.htm here]. The used nucleic acids were designed as follows:

The molecular beacon was purchased from [http://www.eurogentec.com/eu-home.html Eurogentec] and labeled with 6-FAM (fluorophor) and Dabcyl (appropriate quencher for 6-FAM). Preferentially, use a complementary oligonucleotide (target) as well as two enshortened oligonucleotides (split target) for characterizing 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 beacons 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 NAD⁺ 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 closed state and the open state signals is maximal.

• Thermal profile analysis:
  • Prepare a 30 µL reaction mix composed of NAD⁺ 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):

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 NAD⁺ bioassay buffer (F_buffer) and the added molecular beacon (F_closed) as well as the target (F_open) 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 NAD⁺ 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⁺-dependent DNA ligase gene (<partinfo>K525710</partinfo>) 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 (<partinfo>K525710</partinfo>) in 30 mL LB medium containing 20 µg mL^-1 chloramphenicol at 37 °C.
  • Dilute the overnight culture in 100 ml autoinduction medium (20 µg mL^-1 chloramphenicol added) to an OD₆₀₀ = 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 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 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 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 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 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 SDS-PAGE analysis.
  • Determine the protein concentration with a 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 NPI-10 to 1-5 mM. If the eluate contains unspecifically bound proteins maybe a washing step with NPI-10 containing 50 mM or 100 mM imidazole could help to get higher purity.

• Large-scale purification
  • For production of DNA ligase with high yield see chapter affinity chromatography.

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 useful for the NAD⁺ detection so that the AMP moiety has to be removed.

• Mix the purified DNA ligase with deadenylation buffer and incubate for 20 min at 37 °C.
• 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] ([http://www.sartorius.de/index.php?id=156&no_cache=1 Sartorius]) concentrators with a 5000 molecular weight cut-off PES membrane. Repeat the procedure several times by adding DNA ligase buffer (remove of NMN and formed NAD⁺).
• Check the deadenylation process by SDS-PAGE analysis with high resolution. If the deadenylation was successful there should not be a double band indicating both forms of DNA ligase.
• Determine the protein concentration with a Bradford Protein Assay.
• For long-time storage keep the protein at -20 °C.

NAD⁺ bioassay

• Prepare a master mix composed of 250 nM molecular beacon and 250 nM split target (notice that the 5'-end at the nick site has to be phosphorylated) in NAD⁺ bioassay buffer.
• Incubate for 20 min at 37 °C.
• Add 500 ng purified and deadenylated DNA ligase (final concentration 5 ng/µL).
• Monitor the fluorescence with a spectrofluorometer at 37 °C and wait until it reaches equilibrium.
• Add 1 µl NAD⁺ in different final concentrations (e.g. 0, 5, 10, 20, 30, 50, 80, 100, 200, 300, 500, 1000, 10000 nM) filling the reaction mix up to 100 µL, stirr shortly and monitor the fluorescence at 37 °C again.
• Determine the initial enhancement rate of flourescence intensity for each NAD⁺ concentration (in a range of 200 s after NAD⁺ addition).
• For the calibration curve plot the initial velocity versus the NAD⁺ concentration.

Imaging:

• It is a similar procedure to the one mentioned above except that you prepare 500 nM molecular beacon and 600 nM target or split target dissolved in bioassay buffer in a PCR tube.
• After incubation at 37 °C add 1000 ng DNA ligase and possibly NAD⁺ in different concentrations filling the reaction mix up to 200 µL.
• Visualize the fluorescence in a laser scanner or UV transilluminator and document the results.