Team:Tuebingen/Experiments



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> Agarose Gels

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  • cast gels
  • load samples with loading dye
  • run gels at 120 V


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> Expression

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  • pETue-NAGA-Intein was transformed into E.coli BL21(DE3)
  • grow culture to OD_600 = 0.3
  • induce with 0.2 mM IPTG
  • express O/N at room temperature
  • centrifuge at 6000 g for 30 minutes at 4°C
  • resuspend pellet in 10 ml lysis buffer
  • sonicate for a total of 3 minutes (40% amplitude, 1 s intervals)


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> Fluorescence assay

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  • Cells were measured undiluted or in 1:2 dilution in 96 well plates (black, flat bottom)
  • Following excitation and emission wavelengths were used for the different fluorescent proteins:
  • GFP
    • Exc: 488 nm
    • Em: 520 nm
    • Dronpa
      • Exc: 503 nm
      • Em: 535 nm
      • RFP
        • Exc: 584 nm
        • Em: 616 nm
        • Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10
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          > Fluorescent-peptide-coupling Assay

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          • incubate 100 µl of Ni-NTA purified eluate with 10 µl (10 mg/ml, 7,90 mM) peptide solution for 24 or 48 hours at 5°C or room temperature
          • dilute with 110 µl of 2x Laemmli buffer
          • incubate at 95°C for 10 minutes
          • load onto SDS polyacrylamide gel


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          > Fluorescence Microscopy

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            Sample preparation


          • A thin layer of low melting agarose was supplied on glass slides
          • 10 µl Yeast cells grown overnight were inoculated in the solidified agarose
          • samples were used for microscopy after at least 30min incubation at 30° in a humid environment


            • Microscopy Setup


          • We used a Zeiss LSM 710 microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. All pictures were taken with one active fluorescent channel and brightfield channel. Dronpa was observed by using a 514 nm laser (2% power) for excitation, and RFP was observed by using a 633 nm laser (2% power) for excitation


            • Dronpa Illumination


          • Illumination of dronpa was performed in vivo using a 488 nm argon laser at 50% power for off switching and a 405 nm for on switching of fluorescence. The illumination of cells was either performed using continuous illumination for 10 to 20 seconds or by performing a line scan with the illumination laser.


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          > Ni-NTA Purification

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            Pellet

          • centrifugation of the bacterial culture for 10 min at 8000 rpm at 4 °C
          • discard the supernatant
            • Lysis
          • per 1g wet weight of the pellet add 4ml lysis buffer
          • fill up to 0.5 mg/ml with lysozyme
          • incubation on ice for 2 h
          • centrifuge for 30 min at 10000 g at 4 °C
            • Column
          • equilibrate the column with 0.5 ml Ni-NTA (0.25 ml bed volume)
          • add 0.5 ml lysis buffer and slightly centrifuge the buffer through the column, it has to stay wet (do twice)
          • pump the cell lysate (supernatant) for 1 h at ice through the column
            • Wash
          • twice with 1.25ml wash buffer
          • four times with 0.5 ml elution buffer
          • recover the supernatant


          • Load a sample of every step in an SDS gel.


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          > Luciferase Assay

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          • Grow overnight culture of yeast:Induction or repression of luciferase expression by addition of different galactose/glucose/raffinose concentrations for pGal or pSUC promoters.
          • NanoLuc luciferase protocol: add 25 µl cell culture, 25 µl reconstituted luciferase assay reagent and 50 µl PBS in 96 well plate (white, clear bottom)
          • Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay reagent
          • Luciferase activity measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10.


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          > MALDI-TOF mass spectrometry

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            Preparation and trypsin digest:

          • cut bands from SDS polyacrylamide gel
          • cover gel piece with Buffer2 for 30 min to discolor
          • cover with acetonitrile (ACN) for 10 min to dehydrate
          • cover with Buffer1 containing 10 mM DTT to reduce for 45 min
          • cover with Buffer1 containing 55 mM iodoacetamide (IAA) to alkylate for 30 min
          • cover with Buffer1 for 15 min to wash
          • cover with Buffer2 for 10 min to wash
          • cover with 100% ACN for 10 min to dehydrate
          • cover with 20 µl Buffer1 containing 100 ng Trypsin
          • digest for 5 h at 37 °C
          • stop reaction by addition of 2 µl of 10% TFA
          • extract peptides by incubation for 30 minutes


            • Buffer1: 50 mM ammoniumbicarbonat (AB) Buffer2: 70% 50 mM AB, 30% ACN


            Measurement:

          • mix 1 ml of sample with 1 µl of gentisic acid matrix solution
          • pipette unto target
          • wait for crystallisation
          • measure with MALDI-TOF
          • analyse data using FlexAnalysis und FlexControl


            • The result score is calculated as Score = -log10(P).



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          > Purification

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          • centrifuge lysate at 15,000 g for 30 min
          • purify supernatant with Ni-NTA beads (native conditions, gravity flow)
          • elute once with 500 µl elution buffer


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          > SDS-page

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          • Cast gels with composition as described <here> (Link bitte einfügen zu der Zusammensetzung)
          • Load samples with SDS-PAGE loading dye
          • Run gels at 150V until front is run through the gel