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Team:Bielefeld-Germany/Labjournal/Analytics
From 2013.igem.org
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
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
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)]
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
