DNA amount
II.5. W ORKING WITH PROTEINS
II.5.6. D ETECTION OF PROTEINS VIA W ESTERN BLOTTING
Western blotting was applied to detect His6‐ and/or myc‐tagged as well as untagged proteins in various cell lysates and fractions resulting from purification or enrichment steps. In principle, the Western blotting covered two steps. Within the first step, the SDS‐PAGE separated proteins were transferred to a PVDF membrane by following a semi‐dry blot protocol [II.5.6.1]. In the second step, selected proteins or protein tags blotted onto the membranes were immunochemically stained and detected via either chemiluminescence [II.5.6.2] or infrared fluorescence [II.5.6.3].
BL O T T I NG O F P R O T E I N S V I A S E M I‐D R Y B L O T
Before blotting, the SDS‐PAGE gels were incubated in blot buffer on a horizontal shaker with moderate agitation for 15 min. Meanwhile, the Immobilon FL PVDF membranes (Merck Millipore) were activated with methanol for 15 sec and subsequently also incubated in blot buffer. Shortly before blotting, per membrane,
six pieces of filter paper (Gel Blotting Paper GB003, Whatman) were soaked with blot buffer. The filter papers, membranes, and gels were assembled as shown in Figure II‐2 and the proteins were blotted at 20 V for 45‐90 min using a Trans‐Blot SD semi‐dry transfer cell (Bio‐Rad).
IM M U N O C H E M I C A L D E T E C T I ON O F PR O T E I N S U S I NG C H E M I L U M I N E S C E NC E
For the immunochemical detection of tagged and untagged proteins via chemiluminescence, the blotted membranes were incubated in 5 % milk solution for 60 min in order to block nonspecific binding valences on the membranes and thus to reduce background and nonspecific signals. The 5 % milk solution was replaced by dilutions of the respective primary antibodies [II.12.5] in 0.5 % milk solution and the membranes were incubated therein either at room temperature for 1.5‐4 h or at 4 °C overnight. Here, the incubation times depended on the assumed target protein amounts. Excess primary antibodies, which had not bound to the blotted proteins, were removed by three washing steps, each for 10 min, using PBS. Afterwards, the membranes were incubated at room temperature for 1‐4 h in dilutions of the appropriate HRP‐conjugated secondary antibodies [II.12.5] in 0.5 % milk solution.
Finally, the membranes were again washed three times with PBS for 10 min. Please note that all incubation or washing steps described above were done on a horizontal shaker with moderate agitation.
The staining of tagged and untagged proteins, which were recognized by the primary and HRP‐conjugated secondary antibodies, was performed with the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific) according to the manufacturer’s instructions. The resulting chemiluminescence signals were visualized by the Fusion FX7 (Vilber Lourmat) imaging system.
5 % milk solution: 5 % (w/v) powdered milk
in PBS
0.5 % milk solution: 10 % (v/v) 5 % powdered milk solution
in PBS
Immunochemical detection of proteins via infrared (IR) fluorescence was used for the verification of low abundant endogenous AKR1B15 isoforms in biological samples.
In contrast to the detection of proteins via chemiluminescence, the membranes used for the detection via IR fluorescence were blocked through an incubation in IR blocking solution for 60 min. The blocking solution was replaced by appropriate dilutions of monoclonal anti‐AKR1B15 antibodies [II.12.5] in antibody solution. After the incubation in primary antibody dilution solutions at 4 °C overnight, the membranes were washed three times with PBS‐T for 10 min to remove unbound primary antibodies. Afterwards, the membranes were
incubated in appropriate dilutions of the respective IR dye‐conjugated secondary antibodies [II.12.5] in antibody solution for 2‐3 h. Finally, the membranes were washed three times with PBS‐T for 10 min and once more with PBS. Please note that all incubation or washing steps described above were done on a horizontal shaker with moderate agitation and protected from light. In general, membranes were dried between sheets of tissue papers for higher signal intensities and storage.
The detection of IR fluorescence signals was performed with an Odyssey infrared imaging system (LI‐COR) by analyzing both wet and dry membranes.
IR blocking solution: 50 % (v/v) Odyssey Blocking Buffer (PBS)
in PBS (pH 7.4)
antibody solution: 50 % (v/v) Odyssey Blocking Buffer (PBS)
in PBS‐T
II.6. E STABLISHMENT OF MONOCLONAL ANTI ‐ AKR1B15 ANTIBODIES
Monoclonal anti‐AKR1B15 antibodies were generated in cooperation with Dr. med. Elisabeth Kremmer (Service Unit Monoclonal Antibodies of the Helmholtz Zentrum München).
In order to identify peptide sequences within the both human AKR1B15 isoforms which are surface exposed and at the same time possess high immunogenic potential, the protein sequence of AKR1B15.2 was scanned for these features by the technical support of the Peps4LS GmbH. Out of the identified protein regions three epitopes with highest heterogeneity to the human AKR1B10 were selected as targets for the generation of monoclonal antibodies [Table II‐14, Figure III‐4]. These peptides were both synthesized and coupled to ovalbumin or biotin by Peps4LS GmbH.
Table II‐14: Peptides (coupled to ovalbumin, C‐) used for the immunization of Lou/c rats and C57/BL6 mice.
antibody antigenic peptide AKB‐1 C‐PQVNSTNNFHQGPL
AKB‐2 C‐QGFKTGDDFFPKDDKGNMISGKGTF AKB‐3 C‐NRNWRAFDFKEFSHLEDFPFDAEY‐COOH
The production and initial testing of monoclonal antibodies was performed in the group of Elisabeth Kremmer (Service Unit Monoclonal Antibodies, HMGU) according to their standard procedures. In short, a mixture of 50 μg antigenic peptide coupled to ovalbumin (Peps4LS) [Table II‐14], 5 nmol CPG oligonucleotides (TIB MOLBIOL), 500 μl PBS, and 500 μl Freund’s adjuvant was used for the subcutaneous and intraperitoneal immunization of Lou/c rats and C57/BL6 mice. Six weeks after the primary injection, a boost without adjuvant was given. Immune spleen cells of rats and mice were taken and fused with the myeloma cell line P3X63Ag8.653 (CRL‐1580TM, ATCC®) for the generation of monoclonal hybridoma cells.
Supernatants of the hybridoma cultures were initially tested in differential ELISAs, using avidin coated ELISA plates and biotinylated AKR1B15 peptides or biotinylated irrelevant off‐target peptides.
The specificity of monoclonal antibodies which bound to the respective AKR1B15 but not to the off‐target peptides in the initial ELISAs (see VI.3) was further analyzed by Western blotting in our lab. Since the supernatants resulting from the primary hybridoma clones possibly included also nonspecific IgM antibodies, the Western blots for the identification of clones producing IgG antibodies that recognize only the AKR1B15 isoforms (but not other human AKRs) were performed with subclass specific HRP‐conjugated secondary antibodies.
The herein identified AKR1B15 specific hybridoma clones were established by the group of Elisabeth Kremmer (Service Unit Monoclonal Antibodies, HMGU) and the supernatants of the established hybridoma clones were used for the detection of AKR1B15 isoforms.
II.7. S UBCELLULAR LOCALIZATION STUDIES USING
H E L A CELLS AND FLUORESCENCE MICROSCOPY
Confocal fluorescence microscopy was used for the analysis of the subcellular localization of AKR1B15 isoforms overexpressed in HeLa cells.
The day before transient transfection, 4x104 HeLa cells (in 2 ml culture medium per well) were sown on 6‐well plates containing ethanol and flame treated cover slips. The next day, the cells were transiently transfected with plasmids encoding untagged, N‐terminally myc‐
tagged, or C‐terminally myc‐tagged AKR1B15.1, AKR1B15.1 S8R, or AKR1B15.2. For this, the coding sequences of the wild type AKR1B15 isoforms had been cloned into pcDNA3.1(+) (Invitrogen), N‐myc‐pcDNA3 (Ferdinand Haller), and pcDNA4‐myc/His B (Invitrogen) via the HindIII/XhoI, NotI/XhoI, and NotI/HindIII restriction sites, respectively [II.12.4.2]. The plasmids encoding for the AKR1B15.1 S8R mutant were obtained by the mutagenesis of the wild type plasmids [II.12.4.2]. In addition, plasmids encoding for variable long N‐terminal sequences of AKR1B15.1, AKR1B15.2, and AKR1B10 fused to AcGFP were also transiently transfected to HeLa cells in order to analyze the influence of the N‐terminal amino acid sequence of AKR1B15 on its subcellular localization. Here, the coding sequences of the N‐termini were cloned into pAcGFP‐N1 (Clontech) via the HindIII/AgeI restriction sites [Table II‐15, II.12.4.2]. For counterstaining of the endoplasmic reticulum (ER) or cytosol, HeLa cells were co‐transfected with pDsRed2‐ER (Clontech) or pCMV DsRed‐Express2 (Clontech), respectively.
Table II‐15: Plasmid constructs used in subcellular localization studies with AKR1B10 or AKR1B15 N‐termini‐AcGFP fusion proteins.
plasmid N‐terminus
template primers for cloning / mutagenesis
pAcGFP‐N1 ‐
AKR1B15.1 Met1‐Gly26 pET28a(+)‐AKR1B15.1 # 2541 + # 2711 AKR1B15.1 Met1‐Glu30 pET28a(+)‐AKR1B15.1 # 2541 + # 2705 AKR1B15.1 Met1‐Ala38 pET28a(+)‐AKR1B15.1 # 2541 + # 2708 AKR1B15.2 Met1‐Leu30 pET28a(+)‐AKR1B15.2 # 2542 + # 2704 AKR1B15.2 Met1‐Glu58 pET28a(+)‐AKR1B15.2 # 2542 + # 2705 AKR1B15.2 Met1‐Ala66 pET28a(+)‐AKR1B15.2 # 2542 + # 2708 AKR1B10 Met1‐Glu30 pET28a(+)‐AKR1B10 # 2541 + # 2707 AKR1B10 Met1‐Ala38 pET28a(+)‐AKR1B10 # 2541 + # 2708
AKR1B10 K22R Met1‐Ala38 pAcGFP‐AKR1B10 (Met1‐Ala38) # 2921 + # 2922 AKR1B10 P24L Met1‐Ala38 pAcGFP‐AKR1B10 (Met1‐Ala38) # 2919 + # 2920 The detailed amino acid sequences of the different N‐termini can be seen from the alignment of the AKR1B15 isoforms and AKR1B10 in Figure III‐4.
The transfections were carried out as described in II.3.2 via the X‐tremeGENE 9 DNA Transfection Reagent. The cells were incubated at 37 °C and 5 % CO2 for two days in order to express the transfected plasmids. In contrast to the counterstaining of ER and cytosol, the counterstaining of mitochondria was performed prior to the fixation of cells on the cover slips. For this, the culture medium was replaced by 300 nM MitoTracker Orange CMTMRos (Molecular Probes) in serum‐free medium. The cells were incubated therein at culturing conditions for 30 min. Thereby, the MitoTracker Orange CMTMRos cationic dye accumulates in the mitochondria of living cells due to their membrane potential and allows for the permanent staining of mitochondria after fixation.
To prepare objects for microscopy, the cell layers were washed twice with 3 ml/well PBS before they were incubated in 2 ml/well fixation solution at 37 °C, 5 % CO2 for 10 min to fix the cells on the cover slips. After fixation, the cell layers were washed once with 3 ml/well PBS and were then incubated in 2 ml/well permeabilization solution at room temperature for 5 min. For the immunocytochemical staining of proteins or protein tags, the cell layers were washed twice with 3 ml/well PBS before free valences were blocked with 2 ml/well blocking solution for 30‐60 min. The cell layers were washed once with 3 ml/well PBS and were afterwards incubated in 1 ml/well of the respective primary antibody [II.12.5] dilution in blocking solution at room temperature for 1.5‐3 h. Before and after the subsequent incubation in 1 ml/well of the respective secondary antibody [II.12.5] dilution in blocking solution at room temperature for 1‐2 h, the cell layers were washed twice with 3 ml/well PBS.
In contrast to the immunocytochemical staining, cell layers for the subcellular localization analysis of N‐termini‐AcGFP fusion proteins were only washed twice with 3 ml/well PBS after the permeabilization step without any blocking or antibody staining afterwards.
In the end, nuclei were stained by Hoechst 33342 (Molecular Probes), a dye emitting blue fluorescence when bound to the double‐stranded (chromosomal) DNA. For this cell layers were incubated in 1 ml/well Hoechst 33342 dilution (1:5000 in PBS) for 1 min before the cell
layers were once more washed twice with 3 ml/well PBS. Finally, the cover slips were taken out of the wells, dipped into MilliQ‐H2O to reduce salt remains, and mounted on SuperFrost Plus microscope slides (Thermo Scientific) with VectaShield mounting medium (VectorLabs).
The cover slips were fixed with nail polish and the objects were stored at ‐20 °C.
The subcellular localization data were collected and analyzed by confocal fluorescence microscopy using a Zeiss AxioImager Z1/ApoTome confocal microscope with a 63x water immersion objective and a Zeiss AxioCam MRm camera. The recording was controlled and processed by the AxioVision Rel. 4.8 software.
fixation solution: 10 % (v/v) formaldehyde solution (≥ 36.0 %)
in PBS (pH 7.4)
permeabilization solution: 0.5 % (v/v) Triton X‐100
in PBS (pH 7.4)
blocking solution: 3 % (w/v) albumin fraction V
in PBS (pH 7.4)