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Team:Bielefeld-Germany/Labjournal/Analytics
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<h3>Sodium dodecyl sulfate polyacrylamide gel electrophoresis</h3> | <h3>Sodium dodecyl sulfate polyacrylamide gel electrophoresis</h3> | ||
<br><b><u>Pouring the polyacrylamide gel</u></b> | <br><b><u>Pouring the polyacrylamide gel</u></b> | ||
| - | <p>Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED</p> | + | <p>- Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED</p> |
| - | <p>Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel</p> | + | <p>- Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel</p> |
| - | <p>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</p> | + | <p>- 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</p> |
| - | <p>Layer isopropanol on top of the ge</p> | + | <p>- Layer isopropanol on top of the ge</p> |
| - | <p>Leave the separating gel at room temperature for >60 minutes to polymerize</p> | + | <p>- Leave the separating gel at room temperature for >60 minutes to polymerize</p> |
| - | <p>Remove isopropanol and wait until the surface is dry</p> | + | <p>- Remove isopropanol and wait until the surface is dry</p> |
| - | <p>Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form</p> | + | <p>- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form</p> |
| - | <p>Insert comb without getting bubbles stuck underneath</p> | + | <p>- Insert comb without getting bubbles stuck underneath</p> |
| - | <p>Leave the gel at room temperature for >60 minutes to polymerize</p> | + | <p>- Leave the gel at room temperature for >60 minutes to polymerize</p> |
<br> | <br> | ||
<p><i>For storage:</i></p> | <p><i>For storage:</i></p> | ||
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Measuring of mRFP with Tecan Infinite® M200 platereader</p> | Measuring of mRFP with Tecan Infinite® M200 platereader</p> | ||
| - | <p>Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination | + | <p>-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 | + | - 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) | + | - 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 Tecan Infinite® M200 platereader) with following settings: | + | - Measure the fluorescence in a platereader (we used a Tecan Infinite® M200 platereader) with following settings: |
20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm) | 20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm) | ||
| - | Measurement mode: | + | - Measurement mode: |
| - | Excitation: 584 nm | + | Top Excitation: 584 nm |
Emission: 620 nm | Emission: 620 nm | ||
Number of reads: 25 | Number of reads: 25 | ||
Revision as of 20:59, 25 September 2013
Analytics
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
Fluorescence measurements
Measuring of mRFP with Tecan Infinite® M200 platereader
-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 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
