Team:Bielefeld-Germany/Labjournal/Analytics

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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]

For measurement of intracellular NADH concentration. 1) Inoculating an overnight culture (30 mL with 1 mL of pre-culture). 2) Centrifugation (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. 3) Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer. 4) Resuspend pellet in 1 mL PBS buffer. 5) Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm). 6) Centrifugation for 10 min at maximum speed. 7) Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader.

Retrieved from "http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/Analytics"

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 </div>
-<div class="sds_headline">
+<div class="nadh_headline">
-<a name="sds"><span style="color:#ff6600; padding-left=22px">[SDS-PAGE]</span></a>
+<a name="nadh"><span style="color:#ff6600; padding-left=22px">[NADH-Assay]</span></a>
 </div>
-<div class="sds" >
+<div class="nadh" >
+For measurement of intracellular NADH concentration.
+) Inoculating an overnight culture (30 mL with 1 mL of pre-culture).
+) Centrifugation (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.
-<h3>Sodium dodecyl sulfate polyacrylamide gel electrophoresis</h3>