Team:Bielefeld-Germany/Labjournal/Analytics

From 2013.igem.org

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<p> • Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader</p>
<p> • Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader</p>
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<br><b><u>Tecan Infinite® M200 platereader parameters: </u></b>
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<br><b style="padding-left:22px;"><u>Tecan Infinite® M200 platereader parameters: </u></b>
<p> • Sample volume = 100 μL clear supernatant </p>
<p> • Sample volume = 100 μL clear supernatant </p>
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<p> • Concentration calculation by NADH calibration curve</p>
<p> • Concentration calculation by NADH calibration curve</p>
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<div class="hexadecan_headline">
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<a name="hexadecan"><span style="color:#ff6600; padding-left=22px">[Hexadecan Assay]</span></a>
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This assay has been used to measure cell membrane hydrophobicity.
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<a name="tecan"><span style="color:#ff6600; padding-left=22px">[Fluorescence measurements]</span></a>
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<br><b><u>Protocol</u></b>
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<br><b><u>Fluorescence measurements </u></b>
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<p>
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<p> • Inoculate an overnight culture (30 mL with 1 mL of pre-culture) </p>
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Measuring of mRFP with Tecan Infinite® M200 platereader</p>
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<p> • Centrifugate (5 min at 4000 g) of 2 mL overnight culture (OD 4-6)</p>
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<p> • Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer</p>
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<p>-Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
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<p> • Resuspend pellet in 1 mL 0,9% NaCl and measurement of OD600</p>
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<p> • Add x μL of washed cells to 0,9% NaCl for a final volume of 3 mL . Final OD600 should be approximately 0,3 (denoted as A0, calculate exact value)</p>
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- 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)
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<p> • Add 3 mL Hexadecan and vortex for 60 sec</p>
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- Measure the fluorescence in a platereader (we used a Tecan Infinite® M200 platereader) with following settings:
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<p> • Incubation for 15 min</p>
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20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm)
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<p> • Discard the upper organic phase and measure OD600 of the aqueous phase (denoted as A)</p>
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- Measurement mode:
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<p> • Hydrophobicity can be calculated using the equation: affinity [%] = 100 x [1 – (A/A0)]</p>
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Top Excitation: 584 nm
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Emission: 620 nm
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Number of reads: 25
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Manual gain: 100
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Integration time: 20 µs
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<div class="periplasmic_headline">
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<a name="periplasmic"><span style="color:#ff6600;">[Cold osmotic shock]</span></a>
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<h3>Release of periplasmic protein fraction from <i>E. coli</i> by cold osmotic shock</h3>
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<br><b><u>Modified protocol from [[Neu & Heppel (1965)]]</u></b>
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<p> • Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells</p>
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<p> • Discard the entire supernatant</p>
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<p> • Resuspend the cells in ice-cold [[cell fractionating buffer 1]]. The resulting volume should be 1/4 of the former suspension volume</p>
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<p> • Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation</p>
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<p> • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)</p>
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<p> • Discard the entire supernatant</p>
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<p> • Resuspend the cells in ice-cold [[cell fractionating buffer 2]]. The resulting volume should be 1/4 of the former suspension volume</p>
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<p> • Incubate for 10 up to 20 min on ice under regular invertion</p>
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<p> • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)</p>
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<p> • Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using [[Cell fractionating buffer 2.2]] and [[2.3]]</p>
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<p> • If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter</p>
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Latest revision as of 11:32, 26 September 2013







Analytics


[SDS-PAGE]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis


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 ge

- 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 comb 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



[NADH-Assay]

This method has been used for measurement of intracellular NADH concentration.
Protocol:

• Inoculate an overnight culture (30 mL with 1 mL of pre-culture)

• Centrifugate (5 min at 5000 g) of 6-10 mL overnight culture (OD 4-6), adjust volume between different samples for the approximation of the number of cells

• Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer

• Resuspend pellet in 1 mL PBS buffer

• Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm)

• Centrifugation for 10 min at maximum speed

• Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader


Tecan Infinite® M200 platereader parameters:

• Sample volume = 100 μL clear supernatant

• Excitation = 340 nm

• Emission = 460 nm

• Concentration calculation by NADH calibration curve

[Hexadecan Assay]
This assay has been used to measure cell membrane hydrophobicity.
Protocol

• Inoculate an overnight culture (30 mL with 1 mL of pre-culture)

• Centrifugate (5 min at 4000 g) of 2 mL overnight culture (OD 4-6)

• Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer

• Resuspend pellet in 1 mL 0,9% NaCl and measurement of OD600

• Add x μL of washed cells to 0,9% NaCl for a final volume of 3 mL . Final OD600 should be approximately 0,3 (denoted as A0, calculate exact value)

• Add 3 mL Hexadecan and vortex for 60 sec

• Incubation for 15 min

• Discard the upper organic phase and measure OD600 of the aqueous phase (denoted as A)

• Hydrophobicity can be calculated using the equation: affinity [%] = 100 x [1 – (A/A0)]



[Cold osmotic shock]

Release of periplasmic protein fraction from E. coli by cold osmotic shock


Modified protocol from [[Neu & Heppel (1965)]]

• Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells

• Discard the entire supernatant

• Resuspend the cells in ice-cold [[cell fractionating buffer 1]]. The resulting volume should be 1/4 of the former suspension volume

• Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation

• Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)

• Discard the entire supernatant

• Resuspend the cells in ice-cold [[cell fractionating buffer 2]]. The resulting volume should be 1/4 of the former suspension volume

• Incubate for 10 up to 20 min on ice under regular invertion

• Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)

• Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using [[Cell fractionating buffer 2.2]] and [[2.3]]

• If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter




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