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From 2012.igem.org

Contents 1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 1.1 Pouring the polyacrylamide gel 1.2 Preparing the sample 1.3 Running the gel 1.4 Polyacrylamide gel staining with colloidal Coomassie Blue 2 Substrate analytics 2.1 HPLC 2.1.1 Protocol to regenerate the RP-packings: 2.2 Liquid chromatography–mass spectrometry 2.2.1 Sample preparation 2.2.1.1 Substrate preperation 2.2.1.2 Degradation 2.2.1.3 SPE-Column purification 3 Activity measurements

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

This analytical method can be used for separation and identification of proteins according to their electrophoretic mobility. The mobility is a function of length of the molecular weight. Proteins that have identical charge per unit mass due to binding of SDS results in an equal electrophoretic mobility.

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 gel.
• 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 (15 µL protein solution + 5 µL 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 comp 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 (5µl 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.

Polyacrylamide gel staining with colloidal Coomassie Blue

Modified staining protocol from [http://http://newjournal.kcsnet.or.kr/main/j_search/j_abstract_view.htm?code=B021105&qpage=j_search&spage=b_bkcs&dpage=ar Kang et al., 2002].

• agitate the staining solution at 37 °C over night to form the colloids
• After finishing the SDS-PAGE remove gel from gel casting form and tranfer it in to a box.
• Wash the gel with H₂O
• Add 100 mL of the stainig solution to your polyacrylamid gel.
• Incubate the gel in the solution at room temperature over night. Shake the gel continuously during incubation.
• Remove the staining solution
• Incubate the gel in fixer solution for 30 minutes
• Wash the gel with H₂O over night.

Note: Shake the gel continuously during incubation or wash steps.

Substrate analytics

HPLC

High performance liquid chromatography (HPLC), a sort of liquid chromatography, is an analytical method in chemistry, not only to separate substances, but also to identify and quantify these substances using internal standards.

The examined substance, together with a mobile phase, is pumped through a separation column, the stationary phase. You can classify an HPLC according to the principle of separation in normal phase (NP), reverse phase (RP), ion exchange (IEC), size exclusion (SEC) and chiral chromatography. The substance to be tested is injected into the stream of the mobile phase in small amounts. The movement of the sample through the column depends on the nature of the sample on the one hand and the stationary phase on the other hand. If one component of the tested substance interacts strongly with the stationary phase, it remains on the column relatively long. If the interaction of a substance is weak, its velocity to leave the column is higher. The time at which a specific substance elutes is called retention time and is suitable for identification under particular conditions.

• C18 reverse phase column
• Isocratic method: 60 % Acetonitrile
• Flow = 0.5 mL min^-1
• Fluorescence-detection
  • Ex 230 nm, Em 310 nm for Estrogens
  • Ex 275 nm, Em 350 nm for PAH´s
• Column:
  • Eurospher II 100-3 C18p by [http://www.knauer.net/ Knauer]
  • Type of column: Vertex Plus Column
  • Precolum
  • Oven temperature 30 °C
• Software:
  • Clarity (Version 4.0.0.681) by [http://www.dataapex.com/ Data Apex]
• Autosampler:
  • Midas by [http://www.spark.nl/ Spark Holland]
  • Tray cooling: 4 °C
• Pump:
  • L-6200A Intelligent Pump by [http://www.hitachi.com/ Hitachi]
• Fluorescence-Detector:
  • RF-551 by Shimadzu

Protocol to regenerate the RP-packings:

• Flush the column with 20 volumes of water
• Flush the column with 20 volumes of acetonitrile
• Flush the column with 5 volumes of isopropanol
• Flush the column with 20 volumes of heptane
• Flush the column with 5 volumes of isopropanol
• Flush the column with 20 volumes of acetonitrile

Liquid chromatography–mass spectrometry

This method can be used to seperate analyts to analyzed with mass-spectrometry. We used this technique to identify degradation products after the Laccase treatment to our Substrates.

For the LC-ESI-qTOF-MS we had the [http://www.bdal.com/products/lc-ms/o-tof/microtof-q-ii/overview.html micrOTOF-Q II™] by Bruker Daltonics available.

We used this Methode to analyze degradation products of Estradiol, Estron, Ethinyl estradiol and Anthracen.

Sample preparation

Substrate preperation

Our Substrates are soluble in Methanol. We set our standarts into a concentration of 1 mg/ml. The detection limit for the LC-MS showed that a concentration of XXXX for the Substrates Esteron and Estradiol is the highest concentration of detection. Same limit of detection was used for Ethinestradiol and Anthracen for degradation. We only used thoose four Substrates. For all LC-MS preperations we used the T. versicolor Laccases.

Degradation

For the degradation of our Substrates we used following standard reaction:

Material	Volume
Robinson Buffer	425µL
Substrates	25µL
[http://www.sigmaaldrich.com/catalog/product/sigma/53739?lang=de&region=DE Laccase]	50µL
(ABTS	5 µL)

The activation of the T. versicolor Laccase was set into 0,1U in 50µL. ABTS was used to speed up the reaction but we did not use it for all Samples. The reactions was stoped with Methanol after degradation over night. If we used the samples directly after the degradation the samples were transered into HPLC wheels otherwise we stored them a 4°C fridge.

SPE-Column purification

To remove unwanted analytes from the samples we used [http://www.phenomenex.com/Products/SPDetail/Strata/C18-E?returnURL=/Products/Search/SPE C18E-SPE-Columns]. Since we don't know anything about the degradation products the columns were used to make a proof of concept. Therefor following steps were necessary to regenerate the column and solve the products from the column:

• 2 ml (8:2 v/v) water-aceton,
• 2 ml Methanol,
• 2 ml Water,
• pipett Sample to the column,
• 2 ml Water,
• 2 ml (8:2 v/v) water-aceton
• and the elution with 1 ml Methanol

after each step we centrigufed the column in 800g.

Activity measurements

For the measurements [http://www.sigmaaldrich.com/catalog/product/sigma/m0312?lang=de&region=DE 96-well flat bottom microplates] were used. Each well contained a total sample volume of 200 µL respectively. The sample setup was pipetted as follows:

component	concentration
buffer:	100 mM
Laccase	0,1 U
[http://www.sigmaaldrich.com/catalog/product/sigma/a1888?lang=de&region=DE ABTS]	0,1 mM
H2O	ad 200 µL

The [http://www.sigmaaldrich.com/catalog/product/sigma/53739?lang=de&region=DE Laccase of T. versicolor] we bought for standardization was diluted in water so that 140 µL would contain 0,1 U meaning 72 x 10^-5 g of the enzyme. Please check the labjournal if Sodium-Acetate or Briton Robinson Buffer was used, respectively. The ABTS and laccase concentration optimum for standardization (so that the reaction was traceable nicely) was determinated during several experiments. Check the labjournal for further information.

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