From b2324dabd57c057823afa39be394525020a32a65 Mon Sep 17 00:00:00 2001 From: Alexander Ralph Michael Minges Date: Sun, 5 Sep 2010 22:26:37 +0200 Subject: [PATCH] Weiterarbeit am Ergebnisteil; Korrekturen in Material/Methoden --- bachelorarbeit.kilepr | 44 +- bachelorarbeit.pdf | Bin 6606123 -> 7092038 bytes bachelorarbeit.tex | 2 +- img/ergebnisse/fluoreszenz_assay/binding.pdf | Bin 0 -> 52628 bytes img/ergebnisse/fluoreszenz_assay/binding.svg | 1197 ++++++ .../fluoreszenz_assay/binding_inset.pdf | Bin 0 -> 55166 bytes .../fluoreszenz_assay/binding_inset.svg | 1723 +++++++++ img/ergebnisse/fluoreszenz_assay/spektrum.pdf | Bin 0 -> 62703 bytes img/ergebnisse/fluoreszenz_assay/spektrum.svg | 3323 +++++++++++++++++ .../photometrischer_assay/ph65_binding.pdf | Bin 0 -> 58553 bytes .../photometrischer_assay/ph65_binding.svg | 1156 ++++++ .../ph65_spektrum_klein.pdf | Bin 0 -> 67132 bytes .../ph65_spektrum_klein.svg | 1198 ++++++ .../photometrischer_assay/ph75_binding.pdf | Bin 0 -> 51962 bytes .../photometrischer_assay/ph75_binding.svg | 1119 ++++++ .../ph75_spektrum_klein.pdf | Bin 0 -> 66147 bytes .../ph75_spektrum_klein.svg | 1216 ++++++ .../photometrischer_assay/ph85_binding.pdf | Bin 0 -> 58488 bytes .../photometrischer_assay/ph85_binding.svg | 1118 ++++++ .../ph85_spektrum_klein.pdf | Bin 0 -> 66696 bytes .../ph85_spektrum_klein.svg | 1228 ++++++ inc/abkuerzungen.tex | 4 +- inc/ergebnisse.tex | 60 +- inc/material.tex | 26 +- 24 files changed, 13382 insertions(+), 32 deletions(-) create mode 100644 img/ergebnisse/fluoreszenz_assay/binding.pdf create mode 100644 img/ergebnisse/fluoreszenz_assay/binding.svg create mode 100644 img/ergebnisse/fluoreszenz_assay/binding_inset.pdf create mode 100644 img/ergebnisse/fluoreszenz_assay/binding_inset.svg create mode 100644 img/ergebnisse/fluoreszenz_assay/spektrum.pdf create mode 100644 img/ergebnisse/fluoreszenz_assay/spektrum.svg create mode 100644 img/ergebnisse/photometrischer_assay/ph65_binding.pdf create mode 100644 img/ergebnisse/photometrischer_assay/ph65_binding.svg create mode 100644 img/ergebnisse/photometrischer_assay/ph65_spektrum_klein.pdf create mode 100644 img/ergebnisse/photometrischer_assay/ph65_spektrum_klein.svg create mode 100644 img/ergebnisse/photometrischer_assay/ph75_binding.pdf create mode 100644 img/ergebnisse/photometrischer_assay/ph75_binding.svg create mode 100644 img/ergebnisse/photometrischer_assay/ph75_spektrum_klein.pdf create mode 100644 img/ergebnisse/photometrischer_assay/ph75_spektrum_klein.svg create mode 100644 img/ergebnisse/photometrischer_assay/ph85_binding.pdf create mode 100644 img/ergebnisse/photometrischer_assay/ph85_binding.svg create mode 100644 img/ergebnisse/photometrischer_assay/ph85_spektrum_klein.pdf create mode 100644 img/ergebnisse/photometrischer_assay/ph85_spektrum_klein.svg diff --git a/bachelorarbeit.kilepr b/bachelorarbeit.kilepr index b93ce3a..f56bd7c 100644 --- a/bachelorarbeit.kilepr +++ b/bachelorarbeit.kilepr @@ -4,7 +4,7 @@ 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+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +Absorption + + + + + + + + + + + + + + + +-0,4 + + + + + + + + + +-0,2 + + + + + + + + + +0 + + + + + + + + + +0,2 + + + + + + + + + +0,4 + + + + + + + + + +0,6 + + + + + + + + + +0,8 + + + + + + + + + +1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +-0,4 + + + + + + + + + +-0,2 + + + + + + + + + +0 + + + + + + + + + +0,2 + + + + + + + + + +0,4 + + + + + + + + + +0,6 + + + + + + + + + +0,8 + + + + + + + + + +1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +Wellenlänge [nm] + + + + + + + + + + + + + + + +300 + + + + + + + + + +350 + + + + + + + + + +400 + + + + + + + + + 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\acro{CV}{\textit{column volume}, Säulenvolumen} - \acro{dNTP}{desoxyribo-Nukleinsäure-Triphosphat} +% \acro{dNTP}{desoxyribo-Nukleinsäure-Triphosphat} \acro{DTT}{Dithiothreitol} \acro{EBF1}{\textit{EIN3 BINDING FACTOR 1}} \acro{EBF2}{\textit{EIN3 BINDING FACTOR 2}} @@ -25,7 +25,7 @@ \acro{ER}{Endoplasmatisches Retikulum} \acro{ERF1}{\textit{ETHYLENE RESPONSE FACTOR 1}} \acro{ETR1}{\textit{ETHYLENE RESISTANT 1}} - \acro{GR}{\textit{GREEN RIPE}} +% \acro{GR}{\textit{GREEN RIPE}} \acro{HRP}{\textit{horseradish peroxidase}, Meerrettich-Peroxidase} \acro{IDA}{Im\-in\-o\-di\-es\-sig\-säu\-re} \acro{IPTG}{Isopropyl-\textbeta-D-thiogalactopyranosid} diff --git a/inc/ergebnisse.tex b/inc/ergebnisse.tex index c6ddeff..f167263 100644 --- a/inc/ergebnisse.tex +++ b/inc/ergebnisse.tex @@ -52,7 +52,65 @@ Um die Spezifität der Bindung nachzuweisen wurde in weiteren Versuchen zur Elut \centering \includegraphics[width=0.7\textwidth,keepaspectratio=true]{./img/ergebnisse/haeminagarose_assay/haemin_agarose_batch_11_08_10_2_beschriftet.pdf} % haemin_agarose_batch_11_08_10_beschriftet.pdf: 683x621 pixel, 72dpi, 24.09x21.91 cm, bb=0 0 683 621 - \caption[Spezifität der Hämin-Bindung]{Spezifität der Hämin-Bindung; Von links nach rechts: Marker (M), Proteinkonzentrat (K), Durchlauf (D), Waschschritte (W1-3), Elutionen (E1, E2); E1 wurde mit \SI{11,1}{\milli\Molar} freiem Hämin, E2 im Anschluss an E1 mit 2x SDS-Probenpuffer und Erhitzen auf \SI{99}{\celsius} eluiert. Die \ac{RTE1}-Banden sind durch Pfeile markiert. Die durchgängige Hintergrundfärbung von E1 und E2 und insbesondere die starke Färbung im Bereich unterhalb von \SI{15}{\kilo\dalton} wird durch das freie Hämin hervorgerufen. Beim Auftragen wurden die Verdünnungsfaktoren berücksichtigt. } + \caption[Spezifität der Hämin-Bindung]{Spezifität der Hämin-Bindung; Von links nach rechts: Marker (M), Proteinkonzentrat (K), Durchlauf (D), Waschschritte (W1-3), Elutionen (E1, E2); E1 wurde mit \SI{11}{\milli\Molar} freiem Hämin, E2 im Anschluss an E1 mit 2x SDS-Probenpuffer und Erhitzen auf \SI{99}{\celsius} eluiert. Die \ac{RTE1}-Banden sind durch Pfeile markiert. Die durchgängige Hintergrundfärbung von E1 und E2 und insbesondere die starke Färbung im Bereich unterhalb von \SI{15}{\kilo\dalton} wird durch das freie Hämin hervorgerufen. Beim Auftragen wurden die Verdünnungsfaktoren berücksichtigt. } \label{fig:haeminagarose} \end{figure} +\section{Photometrischer Bindungsassay} +\label{sec:ergebnis_photometrie} +Zur Analyse der Bindungseigenschaften von \ac{RTE1} an Hämin wurden drei Messreihen bei unterschiedlichen pH-Werten -- pH 6,5; pH 7,5; pH 8,5 -- durchgeführt. Hierfür wurden Aliquots der gereinigten Proteinlösung mit einem Volumen von jeweils \SI{2,5}{\milli\liter} wie in \ref{sec:nachweis_haemin_bindung_photometrie} beschrieben in die Waschpuffer pH 6,5, pH 7,5 und pH 8,5 umgepuffert. Nach Bestimmung der Proteinkonzentration wurden die Proteinlösungen dann auf eine Konzentration von \SI{1,5}{\micro\Molar} verdünnt und in die Messung eingesetzt. Die Obergrenze der eingesetzten Häminkonzentration von \SI{40}{\micro\Molar} ergab sich durch die Spezifikationen des verwendeten Photometers. Eine \SI{50}{\micro\Molar} Häminlösung weist bereits eine maximale Absorption von über 3 auf, der Obergrenze laut Herstellerangaben. + +\begin{figure}[htbp!] + %\centering + \subfloat[pH 6,5]{\includegraphics[width=0.45\textwidth,keepaspectratio=true]{./img/ergebnisse/photometrischer_assay/ph65_spektrum_klein.pdf}} + \subfloat[pH 7,5]{\includegraphics[width=0.45\textwidth,keepaspectratio=true]{./img/ergebnisse/photometrischer_assay/ph75_spektrum_klein.pdf}}\\ + \subfloat[pH 8,5]{\includegraphics[width=0.45\textwidth,keepaspectratio=true]{./img/ergebnisse/photometrischer_assay/ph85_spektrum_klein.pdf}} + \caption[Differenzspektren des photometrischen Bindungsassays]{Differenzspektren des photometrischen Bindungsassays; Insbesondere bei pH 6,5 (a) und pH 7,5 (b) ist deutlich eine Verschiebung der Absorptionsmaxima bei höheren Häminkonzentrationen hin zu kürzeren Wellenlängen sichtbar.} + \label{fig:spektren_uv_vis} +\end{figure} + +Zur Bestimmung des K$_\text{d}$-Werts der Bindung wurde die Verschiebung der Absorptionsmaxima $\Delta A$ zueinander gegen die größte gemessene Verschiebung normiert und gegen die tatsächlich vorliegende Häminkonzentration $c$ aufgetragen. Letztere wurde über den Absorptionskoeffizienten von Hämin aus den aufgenommenen Pufferspektren bestimmt. Anschließend wurde eine Sättigungsfunktion über eine nichtlineare Regression angepasst: + +\begin{equation} + \Delta A = \frac{a \cdot c}{K_d + c} +\end{equation} + +$a$ sei hierbei die Kapazität. Die entsprechenden Auftragungen und Sättingungsfunktionen sind in Abbildung \ref{fig:fit_uv_vis} dargestellt. + +\begin{figure}[htbp!] + %\centering + \subfloat[pH 6,5]{\includegraphics[width=0.45\textwidth,keepaspectratio=true]{./img/ergebnisse/photometrischer_assay/ph65_binding.pdf}} + \subfloat[pH 7,5]{\includegraphics[width=0.45\textwidth,keepaspectratio=true]{./img/ergebnisse/photometrischer_assay/ph75_binding.pdf}}\\ + \subfloat[pH 8,5]{\includegraphics[width=0.45\textwidth,keepaspectratio=true]{./img/ergebnisse/photometrischer_assay/ph85_binding.pdf}} + \caption[Relative Verschiebung der Absorptionsmaxima]{Relative Verschiebung der Absorptionsmaxima; Bei pH 7,5 und 8,5 ist eine beginnende Sättigung zu erkennen, die Kapaziät liegt hierbei jeweils um 1, die K$_\text{d}$-Werte sind unter Berücksichtigung der Standardabweichung annähernd identisch. Bei pH 8,5 fallen die großen Standardabweichungen der Messwerte, sowie die zu hohe Kapazität auf. Letztere sollte durch die Normierung eigentlich 1 betragen. Darüberhinaus ist keine deutliche Sättigung sichtbar.} + \label{fig:fit_uv_vis} +\end{figure} + +\clearpage +\section{Fluoreszenzspektroskopie} +\label{sec:ergebnis_fluoreszenzspektroskopie} + +Nachdem sich im photometrischen Bindungsassay der kleinste K$_\text{d}$-Wert bei einem pH von 7,5 zeigte, wurde diese Bedingung auch für die Fluoreszenzspektroskopie gewählt. Nach Anregung bei einer Wellenlänge von \SI{280}{\nano\meter} und Abzug des Pufferspektrums ergaben sich die in Abbildung \ref{fig:fluoreszenz_spektrum} dargestellten Emissionsspektren. Es zeigte sich ein deutlicher Quench bei zunehmender Häminkonzentration, der seine volle Ausprägung bei einer Häminkonzentration von \SIrange{20}{40}{\micro\Molar} erreichte. + +\begin{figure}[htbp!] + \centering + \includegraphics[width=0.7\textwidth,keepaspectratio=true]{./img/ergebnisse/fluoreszenz_assay/spektrum.pdf} + % spektrum.pdf: 428x377 pixel, 72dpi, 15.10x13.30 cm, bb=0 0 428 377 + \caption[Emissionsspektrum des Fluoreszenz-Assays]{Emissionsspektrum des Fluoreszenz-Assays nach Abzug des Pufferspektrums; Die Anregung erfolgte bei \SI{280}{\nano\meter}. Es ist ein deutlicher Quench bei zunehmender Häminkonzentration zu erkennen. Der Quench ist bei etwa \SIrange{20}{40}{\micro\Molar} Hämin vollständig.} + \label{fig:fluoreszenz_spektrum} +\end{figure} + +\begin{figure}[htbp!] + \centering + \includegraphics[width=0.7\textwidth,keepaspectratio=true]{./img/ergebnisse/fluoreszenz_assay/binding_inset.pdf} + % spektrum.pdf: 428x377 pixel, 72dpi, 15.10x13.30 cm, bb=0 0 428 377 + \caption[Häminbindung im Fluoreszenz-Assay]{Häminbindung im Fluoreszenz-Assay; Aufgetragen sind die relative Intensität des Fluoreszenzsignals gegen die Häminkonzentration. Letztere wurde aus einer Absorptionsmessung mit Hilfe des Extinktionskoeffizienten ermittelt. Das Inset zeigt eine halblogarithmische Auftragung der Daten. In beiden Auftragungen zeigt sich das Erreichen der Sättigung.} + \label{fig:fluoreszenz_spektrum} +\end{figure} +\clearpage + +\section{\ac{CD}-Spektroskopie} +\label{sec:ergebnis_cd_spektroskopie} + + + diff --git a/inc/material.tex b/inc/material.tex index 88181f2..341bb13 100644 --- a/inc/material.tex +++ b/inc/material.tex @@ -12,7 +12,7 @@ \textbf{Bezeichnung} & \textbf{Hersteller}\\ \midrule Analysen-/Präzisionswaagen & Sartorius, Göttingen\\ - Autoklav & H+P Labortechnik, Oberschleißheim\\ + Autoklav & Thermo Fisher Scientific, Bonn\\ Automatische Pipetten & Gilson, Bad Camberg\\ \ac{CD}-Spektrometer J-715 & JASCO Corp., Gross-Umstadt\\ DNA-Gelkammersystem PerfectBlue & PEQLAB Biotechnologie, Erlangen\\ @@ -290,7 +290,11 @@ \item[Häminlösung (\SI{1,1}{\milli\Molar})] \hfill \\ \SI{1}{\milli\gram} Hämin\\ \SI{50}{\micro\liter} \SI{1}{\Molar} \ce{NaOH}\\ - ad \SI{1,39}{\milli\liter} \ce{dH2O} + ad \SI{1,39}{\milli\liter} \SI{50}{\milli\Molar} \ce{Natriumphosphat} pH 7,5 + \item[Häminlösung (\SI{11}{\milli\Molar})] \hfill \\ + \SI{10}{\milli\gram} Hämin\\ + \SI{50}{\micro\liter} \SI{1}{\Molar} \ce{NaOH}\\ + ad \SI{1,39}{\milli\liter} \SI{50}{\milli\Molar} \ce{Natriumphosphat} pH 7,5 \item[Puffer NP] \hfill \\ \SI{500}{\milli\Molar} Natriumchlorid\\ \SI{10}{\milli\Molar} Natriumphosphat pH 7,5 @@ -434,7 +438,7 @@ Auf die Anode der Blotapparatur wurden zunächst drei Lagen Filterpapier, gefolt Nach Abschluss des Blotvorgangs wurde die Membran entnommen und \SI{1}{\hour} in einer \SI{1}{\percent}igen Caseinlösung in TBS auf einem Rotationsschüttler inkubiert. Es schlossen sich zwei Waschschritte mit TBT und einer mit TBS zu jeweils \SI{10}{\minute} an. -Die Membran wurde dann zusammen mit einer \SI{1}{\percent}igen Caseinlösung in TBS, welche den Anti-His-HRP-Antikörper im Verhältnis 1:10000 enthielt, in Folie eingeschweißt und über Nacht bei \SI{4}{\celsius} auf einem Taumelschüttler inkubiert. Am nächsten Tag erfolgten wiederum zwei Waschschritte mit TBT und einer mit TBS zu jeweils \SI{10}{\minute}. Der gebundene Antikörper ließ sich über die gekoppelte \ac{HRP} mit Hilfe eines Chemilumineszenzreagenzes nachweisen. die \ac{HRP} oxidiert in Anwesenheit von Wasserstoffperoxid das Substrat Luminol, wobei es einer Lichtemission kommt. +Die Membran wurde dann zusammen mit einer \SI{1}{\percent}igen Caseinlösung in TBS, welche den Anti-His-HRP-Antikörper im Verhältnis 1:10000 enthielt, in Folie eingeschweißt und über Nacht bei \SI{4}{\celsius} auf einem Taumelschüttler inkubiert. Am nächsten Tag erfolgten wiederum zwei Waschschritte mit TBT und einer mit TBS zu jeweils \SI{10}{\minute}. Der gebundene Antikörper ließ sich über die gekoppelte \ac{HRP} mit Hilfe eines Chemilumineszenzreagenzes nachweisen. die \ac{HRP} oxidiert in Anwesenheit von Wasserstoffperoxid das Substrat Luminol, wobei es zu einer Lichtemission kommt. Hierfür wurde die Membran in Folie eingeschlagen und mit dem Reagenz überschichtet. Für die Visualisierung wurde ein Lumineszenzdetektor (LAS 4000 mini, Fuji) eingesetzt. @@ -444,7 +448,7 @@ Die Proteinkonzentration in einer Lösung kann über einen \acl{BCA}-Assay ermi Im Rahmen dieser Arbeit wurden Proteinbestimmungen mit dem "`BCA Protein Assay Kit"' durchgeführt. Das \ac{BCA}-Reagenz wurde gemäß den Herstellerangaben angesetzt. Anschließend wurden die in den Tabellen \ref{tab:bca_ansaetze_kalibrierung} und \ref{tab:bca_ansaetze_proben} aufgeführten Ansätze in Halbmikroküvetten pipettiert und gevortext. -Es folgte eine Inkubation bei \SI{37}{\celsius} für \SI{30}{\minute} im Brutschrank, woran die Absorptionsmessung anschloss. Bei der Berechnung der Proteinkonzentration in den Proben ist zu berücksichtigen, dass die Proben für die Kalibriergeraden im Vergleich zu den Proben um den Faktor 10 verdünnt wurden. +Es folgte eine Inkubation bei \SI{37}{\celsius} für \SI{30}{\minute} im Brutschrank, woran die Absorptionsmessung anschloss. Bei der Berechnung der Proteinkonzentration in den Proben ist zu berücksichtigen, dass die Proben für die Kalibriergeraden im Vergleich zu den Proben um den Faktor 10 verdünnt wurden. Es wurden jeweils Dreifachbestimmungen durchgeführt. \begin{table}[htb] \centering @@ -464,10 +468,10 @@ Es folgte eine Inkubation bei \SI{37}{\celsius} für \SI{30}{\minute} im Brutsch \begin{table}[htb] \centering \caption{Pipettierschema für \ac{BCA}-Proben} -\begin{tabular}{rrrrrrr} +\begin{tabular}{rr} \toprule -\textbf{Probe [\si{\micro\liter}]} & 10 & 10 & 10 & 10 & 10 & 10 \\ -\textbf{\ac{BCA}-Reagenz [\si{\micro\liter}]} & 990 & 989 & 988 & 987 & 986 & 985 \\ +\textbf{Probe [\si{\micro\liter}]} & 10 \\ +\textbf{\ac{BCA}-Reagenz [\si{\micro\liter}]} & 990 \\ \bottomrule \end{tabular} \label{tab:bca_ansaetze_proben} @@ -486,7 +490,7 @@ Bei Häminen handelt es sich um Komplexverbindungen der Häme, wobei das Eisenio \label{fig:haemin_struktur} \end{figure} -Für den Nachweis einer Häminbindung durch \ac{RTE1} kamen vier unterschiedliche Methoden zum Einsatz: Eine Affinitätschromatographie mit Hämin-Agarose, ein Photometrischer Nachweis über die Zugabe von freiem Hämin, der Nachweis über einen Tyrosin-Quench bei Zugabe von freiem Hämin sowie der Nachweis einer Sekundärstrukturänderung bei Häminzugabe über eine \ac{CD}-Spektroskopie. +Für den Nachweis einer Häminbindung durch \ac{RTE1} kamen vier unterschiedliche Methoden zum Einsatz: Eine Affinitätschromatographie mit Hämin-Agarose, ein Photometrischer Nachweis über die Zugabe von freiem Hämin, der Nachweis über einen Tyrosin-Quench bei Zugabe von freiem Hämin sowie der Nachweis einer Sekundärstrukturänderung bei Häminzugabe über eine \ac{CD}-Spektroskopie. Für jede Methode kam jeweils frisch präpariertes \ac{RTE1} zum Einsatz. \subsubsection{Affinitätschromatographie mit Hämin-Agarose} \label{sec:nachweis_haemin_bindung_affinitaet} @@ -494,14 +498,14 @@ Beim affinitätschromatographischen Nachweis wurde ein Säulenmaterial eingesetz \SI{50}{\micro\liter} Hämin-Agarose-Suspension wurden in ein \SI{1,5}{\milli\liter} Reaktionsgefäß gegeben und drei Mal mit jeweils \SI{150}{\micro\liter} NP-Puffer mit \SI{0,003}{\percent} Fos-Cholin$^\text{\textregistered}$ 16 im Thermoschüttler bei \SI{20}{\celsius} unter Schütteln äquilibriert. Zum Sedimentieren der Agarose-Beads wurden die Reaktionsgefäße \SI{10}{\second} bei \SI{2000}{\g} zentrifugiert. -Nach dem Äquilibrieren wurden \SI{50}{\micro\liter} Proteinlösung auf die Beads gegeben und für \SI{30}{\minute} bei \SI{20}{\celsius} im Thermoschüttler unter Schütteln inkubiert. Es schlossen sich drei Waschschritte mit jeweils \SI{500}{\micro\liter} NP-Puffer versetzt mit \SI{0,003}{\percent} Fos-Cholin$^\text{\textregistered}$ 16 an. Hierbei wurden die Reaktionsgefäße wiederum unter Schütteln für \SI{10}{\minute} bei \SI{20}{\celsius} inkubiert. Die Elution erfolgte durch Zugabe von \SI{50}{\micro\liter} 2x SDS-Probenpuffer und Erhitzen auf \SI{99}{\celsius} für \SI{5}{\minute} oder durch Zugabe einer \SI{11,1}{\milli\Molar} Hämin-Lösung. %Als Negativkontrolle kam eine \ac{BSA}-Lösung in gleicher Konzentration wie die \ac{RTE1}-Probe zum Einsatz. +Nach dem Äquilibrieren wurden \SI{50}{\micro\liter} Proteinlösung auf die Beads gegeben und für \SI{30}{\minute} bei \SI{20}{\celsius} im Thermoschüttler unter Schütteln inkubiert. Es schlossen sich drei Waschschritte mit jeweils \SI{500}{\micro\liter} NP-Puffer versetzt mit \SI{0,003}{\percent} Fos-Cholin$^\text{\textregistered}$ 16 an. Hierbei wurden die Reaktionsgefäße wiederum unter Schütteln für \SI{10}{\minute} bei \SI{20}{\celsius} inkubiert. Die Elution erfolgte durch Zugabe von \SI{50}{\micro\liter} 2x SDS-Probenpuffer und Erhitzen auf \SI{99}{\celsius} für \SI{5}{\minute} oder durch Zugabe einer \SI{11}{\milli\Molar} Hämin-Lösung. %Als Negativkontrolle kam eine \ac{BSA}-Lösung in gleicher Konzentration wie die \ac{RTE1}-Probe zum Einsatz. \subsubsection{Photometrie} \label{sec:nachweis_haemin_bindung_photometrie} -Häm-bindende Proteine zeigen, wenn ein Häm gebunden ist, häufig ein charakteristisches Absorptionsmaximum im so genannten Soret-Band um \SI{400}{\nano\meter}. Außerdem ist hierbei in vielen Fällen eine Rotverschiebung des Soret-Peaks zu beobachten, da das Protein bzw. der Protein-Häm-Komplex unterschiedliche Absorptionsmaxima aufweisen \citep{duncan_heme-binding_1999}. Die Bindung von Hämin an ein Protein kann also durch Beobachtung der Absorptionsmaxima im Soret-Band bzw. ihrer relativen Verschiebung zueinander bestimmt werden. +Häm-bindende Proteine zeigen, wenn ein Häm gebunden ist, häufig ein charakteristisches Absorptionsmaximum im so genannten Soret-Band um \SI{400}{\nano\meter}. Außerdem ist hierbei in vielen Fällen eine Blauverschiebung des Soret-Peaks zu beobachten, da das Protein bzw. der Protein-Häm-Komplex unterschiedliche Absorptionsmaxima aufweisen \citep{duncan_heme-binding_1999}. Die Bindung von Hämin an ein Protein kann also durch Beobachtung der Absorptionsmaxima im Soret-Band bzw. ihrer relativen Verschiebung zueinander bestimmt werden. Hierfür wurde \ac{RTE1} nach der Reinigung über eine PD10-Säule in Waschpuffer pH 7,5 ohne Imidazol umgepuffert. Die Proteinlösung wurde dann mit Waschpuffer soweit verdünnt, dass in einem Endvolumen von \SI{500}{\micro\liter} eine Proteinkonzentration von \SI{1,5}{\micro\Molar} vorlag und in eine Quarzglasküvette überführt. Anschließend wurde ein Absorptionsspektrum im Bereich von \SIrange{300}{500}{\nano\meter} aufgenommen. Hämin wurde hinzutitriert, so dass die in Tabelle \ref{tab:haemin_konzentration_photometrie} aufgeführten Endkonzentrationen erreicht wurden. Alle Messungen erfolgten bei \SI{25}{\celsius}, je Messreihe wurden sechs Spektren aufgenommen. -Zusätzlich wurden Pufferspektren aufgenommen, sowohl ohne als auch mit Hämin-Zugabe. Aus den Pufferspektren wurden dann über den Exktionktionskoeffizienten (Datenblatt des Herstellers) die tatsächliche Hämin-Konzentration in der Küvette bestimmt. Die Auswertung erfolgte über die Differenzspektren. +Zusätzlich wurden Pufferspektren aufgenommen, sowohl ohne als auch mit Hämin-Zugabe. Zur Ermittlung der tatsächlich in der Küvette vorliegenden Häminkonzentration wurde die Küvette mit \SI{500}{\micro\liter} \SI{1}{\Molar} \ce{NaOH} befüllt, Häminstammlösung hinzutitriert und Absorptionsspektren aufgenommen. Aus diesen konnte mit Hilfe des Extinktionskoeffizienten von Hämin (Datenblatt des Herstellers) die Konzentration in der Küvette bestimmt werden. %Aus den Pufferspektren wurden dann über den Exktionktionskoeffizienten (Datenblatt des Herstellers) die tatsächliche Hämin-Konzentration in der Küvette bestimmt. Die Auswertung erfolgte über die Differenzspektren. \begin{table}[ht] \centering

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