Recombinant wild type short isoform QSOX1b (herein referred to as wt QSOX) and SxxC mutant inactive enzyme (herein referred to as C452S QSOX) were obtained as described [23 (link)]. Briefly, competent AD494 E. coli cells (Novagen) were transformed with pET32a-wt QSOX1b [24 (link)] or pET32a-C452S QSOX1b [23 (link)] by electroporation or heat shock and expression was induced by 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) overnight at 20°C. Cells were lysed by French press in native buffer (50 mM phosphate buffer pH 8.0, 500 mM sodium chloride) containing 10 mM imidazole and 50 μM FAD, and the proteins were purified in nickel-agarose beads (Qiagen). Elution buffer consisted of 250 mM imidazole in native buffer. Recombinant proteins were dialyzed against PBS and stored in 50 mM phosphate buffer pH 7.5, containing 1 mM EDTA and 10% glycerol [11 (link)]. Proteins were stored at −20 °C or −80 °C.
Nickel agarose beads
Manufactured by Qiagen
Nickel agarose beads are a type of affinity chromatography resin used for the purification of histidine-tagged recombinant proteins. The nickel ions immobilized on the agarose matrix can selectively bind to the histidine tags present on the target proteins, allowing for their capture and subsequent elution.
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Lab products found in correlation
Bradford assay, by Bio-Rad (1 mentions)
Colloidal blue, by Thermo Fisher Scientific (1 mentions)
Sonicator 3000, by Bioventus (1 mentions)
Phosphorimaging, by Fujifilm (1 mentions)
Tnt reticulocyte lysate system, by Promega (1 mentions)
Tritc conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Centricon, by Merck Group (1 mentions)
7 protocols using nickel agarose beads
1
Recombinant QSOX1b Protein Purification
2
Identification of PCNA-interacting Proteins
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The SH-SY5Y cytosol was kept in isolation buffer for 30 min at 4°C. Following centrifugation at 12,000 g for 20 min, the supernatants were incubated with anti-PCNA antibody to immunoprecipitate PCNA-interacting proteins. The eluted immunoprecipitates were identified by LC-MS/MS or loaded onto 12% SDS-PAGE gels followed by Western blotting using anti-caspase-9 antibody as the primary antibody.
Using the pET system as indicated, wild-type or mutated recombinant His-PCNA proteins were expressed in Escherichia coli BL21 (DE3) cells. The His-tagged fusion proteins were purified with nickel-agarose beads (QIAGEN) according to the manufacturer’s instructions. Approximately 50 μg His-PCNA protein were incubated overnight at 4°C with the lysates obtained from HeLa. Following incubation, nickel-agarose beads were added to capture the recombinant protein and the proteins that bound to it. The PCNA-interacting proteins were identified by Western blot.
Using the pET system as indicated, wild-type or mutated recombinant His-PCNA proteins were expressed in Escherichia coli BL21 (DE3) cells. The His-tagged fusion proteins were purified with nickel-agarose beads (QIAGEN) according to the manufacturer’s instructions. Approximately 50 μg His-PCNA protein were incubated overnight at 4°C with the lysates obtained from HeLa. Following incubation, nickel-agarose beads were added to capture the recombinant protein and the proteins that bound to it. The PCNA-interacting proteins were identified by Western blot.
3
Recombinant Protein Purification and Binding
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Domains for recombinant protein production were cloned into the pET 28a vector in-frame to an N-terminal 6x HIS epitope. His epitope–tagged proteins were manufactured in Escherichia coli BL21(DE3). Following sonication (Misonix Sonicator 3000) in 3 mls ice-cold buffer / 50 ml bacterial culture (150 mM NaCl, 2.7 mM KCl, Na2HPO4, KH2PO4, 20 mM imidazole, 10 mM β-mercaptoethanol), proteins were purified onto 50 ul of Nickel- agarose beads (Qiagen) by 3 hours rolling at 4C. Beads were washed in 10 x 1 ml of the same buffer. Protein yields were determined by Bradford assay (Bio-Rad) and relative protein integrity and purity was determined by SDS-PAGE and Colloidal Blue staining (Invitrogen). Purified recombinant protein was incubated with 10 ul nickel beads in 1 ml of buffer for 2 hours at 4°C with in vitro translated DLL4 proteins made using the TNT-coupled reticulocyte in vitro translation system (Promega). Beads were washed x10 with 1 ml of buffer. Proteins were separated by SDS-PAGE and associated proteins were detected by Western blot.
4
Detecting Ubiquitin Conjugates by IMAC and In Vitro Ubiquitination Assay
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Cells were lysed in buffer containing 6 M guanidinium–HCl and protein–ubiquitin conjugates were captured by immobilized metal affinity chromatography (IMAC) on Nickel-Agarose beads (Qiagen) and washed in 8 M urea. After release from the beads conjugates were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblotting. The antibodies used are listed in Supplementary Experimental Procedures .
In vitro ubiquitination assays were performed as described elsewhere (33 (link)). Briefly, 35S-labelled ELK-1 was generated by cell-free expression using the coupled TNT reticulocyte lysate system (Promega). After removal of unincorporated 35S-methionine by gel filtration (Micro-Spin, BioRad), radio-labelled ELK-1 was incubated with UBQ (10 μg), rE1 (500 ng), rE2 (UBCH5; 500 ng), ATP (4 mM), DTT (1 mM), ubiquitin aldehyde (5 μM) and HeLa Nxt (15–30 μg) for 1 h at 30°C. Reactions were resolved on 8% SDS-PAGE gels, dried and visualized by phosphor-imaging (Fujifilm).
In vitro ubiquitination assays were performed as described elsewhere (33 (link)). Briefly, 35S-labelled ELK-1 was generated by cell-free expression using the coupled TNT reticulocyte lysate system (Promega). After removal of unincorporated 35S-methionine by gel filtration (Micro-Spin, BioRad), radio-labelled ELK-1 was incubated with UBQ (10 μg), rE1 (500 ng), rE2 (UBCH5; 500 ng), ATP (4 mM), DTT (1 mM), ubiquitin aldehyde (5 μM) and HeLa Nxt (15–30 μg) for 1 h at 30°C. Reactions were resolved on 8% SDS-PAGE gels, dried and visualized by phosphor-imaging (Fujifilm).
5
Polyclonal Antibody Production against NET3B
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In order to make a polyclonal antibody against NET3B, DNA corresponding to amino acid residues 157–215 of NET3B was cloned into pGAT4 plasmid. This resulted in the incorporation of an N-terminal His tag into the expressed protein. The recombinant protein was generated in Escherichia coli (Rosseta 2, Novagen) and purified using nickel agarose beads (Qiagen). Polyclonal antibodies were raised in mice as described by Deeks et al. (2012) (link). The specificity of the antiserum was tested on a western blot using total protein extract from two-week-old Arabidopsis seedlings. Immunofluorescence with freeze shattering was performed as described by Zhang et al. (2013) (link). Antibodies were diluted and used at 1:100 for NET3B and 1:500 for BIP2 (Agrisera), followed by secondary antibody incubation with TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch).
6
Plasmid Preparation and Protein Purification
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The plasmids used in this study are listed in Table 1 . Human embryonic kidney 293 cells (HEK 293) and Abelson murine leukemia virus induced, macrophage-like cells from BALB/c mice (RAW264.7) were purchased from ATCC. Both cell lines were grown in DMEM (Thermo Scientific), supplemented with 10% FBS (Atlanta Biologicals) and 100 μg/ml penicillin/streptomycin (Sigma) in 5% CO2. nleB1 (EHEC EDL933), sseK1, and sseK2 (Salmonella enterica ATCC 14028) were cloned into pET42a. A human OGT substrate peptide (KKKYPGGSTPVSSANMM) was fused to the N-terminus of GAPDH and expressed in pET28a. Human FADD was expressed in pET15a. Plasmids were transformed into E. coli BL21 (DE3). Protein overexpression was performed for 4 h at 37°C upon induction with 0.5 mM IPTG. His-tagged proteins were purified using nickel agarose beads (Qiagen) as described previously (El Qaidi et al., 2017 (link)). Salmonella mutants were constructed using lambda red recombination (Datsenko and Wanner, 2000 (link)).
7
Purification and Characterization of Chlamydia Pgp3
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