Team:Aachen/Notebook/Protocols/Analytical methods

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== Bradford assay ==
== Bradford assay ==
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This assay is used for the determination of protein concentration of a sample.  
+
This assay is used for the determination of the protein concentration in a sample.  
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* mix Bradford solution with ddH{{sub|2}}O in a ratio of 1:4
+
* mix the Bradford solution with ddH{{sub|2}}O in a ratio of 1:4
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* prepare about 10 solutions 1 mL each between 125–1,000 μg/mL BSA for a standard curve
+
* prepare about 10 solutions 1 mL, each between 125–1,000 μg/mL BSA for a standard curve
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* use XX/XXX µg/mL BSA as a blank
+
* use pure Bradford solution as a blank
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* mix equal amounts of BSA and samples that concentration is to be known (1-3 µL) with 1 mL of 1x Bradford solution, vortex and incubate 5 min. at room temperature
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* mix equal amounts of BSA and samples with unknown concentrations (1-3 µL) with 1 mL of 1x Bradford solution, vortex and incubate for 5 min. at room temperature
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* measure OD at spectrophotometer at 595 nm
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* measure the OD with a spectrophotometer at 595 nm
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* build a graph from the date of BSA linear range measurement with a concentration against OD and count the concentration of your samples.
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* build a standard curve within the linear range of the BSA data (concentration against OD)
 +
* derive the concentration of your samples from the calibration curve
== Measurement of fluorescence ==
== Measurement of fluorescence ==

Revision as of 14:43, 13 October 2014

Analytical methods

Agarose gel electrophoresis

Separation of DNA or RNA

  1. take 5µl of the PCR product
  2. mix with 1µl loading dye
  3. apply onto agarose gel together with a marker
  4. run at 120 mA for 40 minutes for a full gel


SDS-PAGE

Cell preparation

  • lysis of cell pellet in lysis buffer
  • centrifuge for 15 min at 13.000 rpm
  • mix the supernatant with 2x lammli buffer with β-mercaptoethanol
  • denatured for 5 min at 95 °C
  • sample to the gel

For some SDS-PAGEs, we used BioRad ready made gels.

The recipe of the self-made SDS is as follows:


1.5x Buffer

  • 1.5 M Tris-Cl pH = 8.8
  • in 1 L is 40 ml 10 % SDS


Gels

0.75 mm 12 % RUNNING Gel 1 mm 4 % STACKING Gel
1x 2x 4x 1x 2x 4x
H2O 1.65 mL 3.3 mL 6.6 mL 1.5 mL 3 mL 6 mL
1.5x Gel Buffer 1.3 mL 2.6 mL 5.2 mL 0.65 mL 1.3 mL 2.6 mL
30 % Acrylamide (37.5:1) 2 mL 4 mL 8 mL 0.325 mL 0.65 mL 1.3 mL
10 % APS 50 µL 100 µL 200 µL 25 µL 50 µL 100 µL
TEMED 10 µL 20 µL 40 µL 5 µL 10 µL 20 µL


Run gel

  • apply the prepared samples together with a protein marker on the gel
  • run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V


Bradford assay

This assay is used for the determination of the protein concentration in a sample.

  • mix the Bradford solution with ddH2O in a ratio of 1:4
  • prepare about 10 solutions 1 mL, each between 125–1,000 μg/mL BSA for a standard curve
  • use pure Bradford solution as a blank
  • mix equal amounts of BSA and samples with unknown concentrations (1-3 µL) with 1 mL of 1x Bradford solution, vortex and incubate for 5 min. at room temperature
  • measure the OD with a spectrophotometer at 595 nm
  • build a standard curve within the linear range of the BSA data (concentration against OD)
  • derive the concentration of your samples from the calibration curve

Measurement of fluorescence

The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.

  • volume of sample in each well: 100µl
  • measure GFP fluorescence at an excitation wavelength of 496 nm and an emission wavelength at 516 nm


Measurement of optical density

Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.


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