rabbit polyclonal anti-histag antibody  (Sangon Biotech)

Journal: Biochimie

Article Title: Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

doi: 10.1016/j.biochi.2019.06.010

Figure Lengend Snippet: (A & B) Whole cells extracts (WCE) were fractionated into the following fractions: periplasm and cytoplasm (PP + CP), whole membrane (WM), inner-membrane (IM) and outer-membrane (OM). The localization of proRgpB was determined by Western blotting using anti-RgpB polyclonal antibody. (C) ProRgpB6His from the ΔPorN mutant strain was purified by affinity chromatography on Ni-Sepharose and final products were analyzed by SDS-PAGE and Western blotting with anti-RgpB, anti-6His and anti-RgpBCTD antibodies, and by active-site labeling using the specific biotinylated probe, BiRK. N-terminal amino acid sequences of proteins indicated by arrows were determined by automated Edman-degradation and mass spectrometry analysis. (D) The amino acid sequence of preproRgpB with individual domains coded: lowercase: signal sequence; BLUE: N-terminal pro-domain (PD); RED: catalytic domain; GREEN: Ig-like domain; BLACK: C-terminal domain (CTD). R126 and R229 of the pro-domain and sequences identified by the Edman degradation of indicated (arrows) bands are underlined. Double underlined is a RgpBCTD-derived peptide used to obtain rabbit polyclonal anti-RgpBCTD. Residues mutated in the CTD; to Arg (S664) and to Lys (R700, R701 and R708) are highlighted (see Fig 6).

Article Snippet: The membranes were then incubated with a rabbit polyconal anti-RgpB antibody, a rabbit polyclonal anti-HisTag antibody (GenScript) or a rabbit polyclonal anti-RgpBCTD antibody (custom pAb by Genscript; raised against synthetic peptide RVATAKNRMC) and with the corresponding secondary antibody (anti-rabbit IgG-peroxidase conjugate (Sigma) and anti-mouse IgG-alkaline phosphatase conjugate (Sigma)).

Techniques: Membrane, Western Blot, Mutagenesis, Purification, Affinity Chromatography, SDS Page, Labeling, Mass Spectrometry, Sequencing, Derivative Assay

Journal: Biochimie

Article Title: Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

doi: 10.1016/j.biochi.2019.06.010

Figure Lengend Snippet: ProRgpB6His (0.4 mg/ml) was incubated with the (A) whole cell extract (WCE) of P. gingivalis gingipain-deficient strain ΔK/ΔRAB-A or (B) with the whole bacteria diluted in RPMI media (B-RPMI), alone or in the presence of DTT (5 mM) or 4,4’-Dithiodipyridine disulphide (2 mM). At various time points, aliquots were removed to follow the maturation of the zymogen by Western blotting with pAb anti-RgpB. RgpB activity was also recorded during incubation with (black circle) or without DTT (open circle) using the chromogenic substrate L-BAPNA (panel C: WCE; panel D: intact bacterial cells in B-RPMI). Results are expressed as percentage of activity of the mature RgpB incubated under the same condition (n = 3). (E) The active form of RgpB obtained during the incubation with the bacterial suspension was identified by active-site labelling using the biotinylated probe BiRK. Mature RgpB bearing a C-terminal HisTag (lane M) was used as control.

Article Snippet: The membranes were then incubated with a rabbit polyconal anti-RgpB antibody, a rabbit polyclonal anti-HisTag antibody (GenScript) or a rabbit polyclonal anti-RgpBCTD antibody (custom pAb by Genscript; raised against synthetic peptide RVATAKNRMC) and with the corresponding secondary antibody (anti-rabbit IgG-peroxidase conjugate (Sigma) and anti-mouse IgG-alkaline phosphatase conjugate (Sigma)).

Techniques: Incubation, Bacteria, Western Blot, Activity Assay, Suspension, Control

Journal: Biochimie

Article Title: Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

doi: 10.1016/j.biochi.2019.06.010

Figure Lengend Snippet: ProRgpB6His (0.4 mg/ml) was incubated at room temperature in gingipain activity buffer with/without 5 mM DTT supplementation. At various time points, aliquots were removed and subjected to (A) SDS-PAGE electrophoresis, or (B) Western blot analysis with pAb anti-RgpB, mAb anti-6HisTag and pAb anti-CTD domain. (C) Active-site labelling using the biotinylated probe BiRK to detect active forms of partially processed proRgpB. Mature RgpB bearing a C-terminal HisTag (Veillard et al, 2015) was used as a control (lane M). (D) The activity of RgpB at each time point was determined with the chromogenic substrate L-BAPNA. Results are expressed as percentage of activity of the native RgpB incubated in the same condition (n = 3).

Article Snippet: The membranes were then incubated with a rabbit polyconal anti-RgpB antibody, a rabbit polyclonal anti-HisTag antibody (GenScript) or a rabbit polyclonal anti-RgpBCTD antibody (custom pAb by Genscript; raised against synthetic peptide RVATAKNRMC) and with the corresponding secondary antibody (anti-rabbit IgG-peroxidase conjugate (Sigma) and anti-mouse IgG-alkaline phosphatase conjugate (Sigma)).

Techniques: Incubation, Activity Assay, SDS Page, Electrophoresis, Western Blot, Control

Journal: Biochimie

Article Title: Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

doi: 10.1016/j.biochi.2019.06.010

Figure Lengend Snippet: (A) Inhibitors EDTA (5 mM), TLCK (5 mM), Leupeptin (5 mM) or 4,4’-Dithiodipyridine disulphide (2 mM) were added to bacterial cultures adjusted to OD600=1.5. After centrifugation, bacteria were washed once and diluted PBS-containing inhibitors and lysed by ultrasonication. ProRgpB6His present in the intracellular compartments was analyzed by Western blot with anti-RgpB. (B) ProRgpB6His in the T9SS-deficient ΔporN background was purified by affinity chromatography with 2 mM 4,4’-Dithiodipyridine disulphide added at each purification step. Final products were analyzed by SDS-PAGE, by Western blotting and by active-site labeling using the biotinylated probe BiRK. N-terminal sequences were determined by automated Edman-degradation as indicated. (C) ProRgpB6His (0.4 mg/ml) was incubated at room temperature in gingipain activity buffer supplemented with 10 mM DTT. At various times points, aliquots were removed to follow the proteolytic autoprocessing of the enzyme by SDS-PAGE and Western blotting. The active forms of processed proRgpB were identified by active-site labeling using the biotinylated probe BiRK. Mature RgpB bearing a C-terminal HisTag (lane M) was used as positive control. The incubation process was repeated in the presence of each of the three active gingipains (RgpB, HRgpA and Kgp) at a molar ratio 1:100. Incubations for 96 h in presence of TLCK were used as negative control (Ctl). (D) Alternatively, the processing in presence of active RgpB in the reduced conditions was followed by Native-PAGE electrophoresis.

Article Snippet: The membranes were then incubated with a rabbit polyconal anti-RgpB antibody, a rabbit polyclonal anti-HisTag antibody (GenScript) or a rabbit polyclonal anti-RgpBCTD antibody (custom pAb by Genscript; raised against synthetic peptide RVATAKNRMC) and with the corresponding secondary antibody (anti-rabbit IgG-peroxidase conjugate (Sigma) and anti-mouse IgG-alkaline phosphatase conjugate (Sigma)).

Techniques: Centrifugation, Bacteria, Western Blot, Purification, Affinity Chromatography, SDS Page, Labeling, Incubation, Activity Assay, Positive Control, Negative Control, Clear Native PAGE, Electrophoresis

Journal: Biochimie

Article Title: Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

doi: 10.1016/j.biochi.2019.06.010

Figure Lengend Snippet: (A) ProRgpB6His-ΔCTD (0.4 mg/ml) was incubated alone or with mature RgpB (molar ratio 1:100) at room temperature in gingipain activity buffer supplemented with 10 mM DTT. At various time points, aliquots were removed to determine proteolytic processing of ProRgpB6His-ΔCTD by SDS-PAGE electrophoresis, Western blot with anti-RgpB or anti-HisTag and active-site labelling with the biotinylated probe BiRK. (B) Time-dependent processing of proRgpB6His-ΔCTD by the mature RgpB was analyzed by 12% native gel electrophoresis. Mature RgpB bearing a C-terminal HisTag (lane M) was used as positive control. Incubations for 96 h in presence of TLCK were used as a negative control (Ctl)

Article Snippet: The membranes were then incubated with a rabbit polyconal anti-RgpB antibody, a rabbit polyclonal anti-HisTag antibody (GenScript) or a rabbit polyclonal anti-RgpBCTD antibody (custom pAb by Genscript; raised against synthetic peptide RVATAKNRMC) and with the corresponding secondary antibody (anti-rabbit IgG-peroxidase conjugate (Sigma) and anti-mouse IgG-alkaline phosphatase conjugate (Sigma)).

Techniques: Incubation, Activity Assay, SDS Page, Electrophoresis, Western Blot, Nucleic Acid Electrophoresis, Positive Control, Negative Control

Journal: Biochimie

Article Title: Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

doi: 10.1016/j.biochi.2019.06.010

Figure Lengend Snippet: (A) P. gingivalis strains ΔK/ΔRAB, W83 and ΔPorU at early stationary phase were adjusted to OD600=1.5 and centrifuged for 10 min at 5,000 × g. Bacteria were resuspended in RPMI media complemented with 5 mM DTT and 50 μM of gingipain-specific inhibitors KYT-1 and KYT-36. After 15 min, proRgpB6His (0.4 mg/ml) was added and incubated at room temperature. At various time, aliquots were removed to follow the maturation of the enzyme by Western blotting with the pAb anti-RgpB. The final products obtained after 96 hours of incubation were also analysed by Western blotting with mAb anti-6His and pAb anti-RgpBCTD domain. Mature RgpB bearing a C-terminal HisTag (lane M) was used as a control. (B) Comparison of RgpB-WT (expressed in the W83 strain) and RgpBC473A (expressed in the ΔRgpA strain) maturation by Western blot analysis with pAb anti-RgpB (WC, whole culture; WCE, whole cell extract; M, media; OMV, outer-membrane vesicles)

Article Snippet: The membranes were then incubated with a rabbit polyconal anti-RgpB antibody, a rabbit polyclonal anti-HisTag antibody (GenScript) or a rabbit polyclonal anti-RgpBCTD antibody (custom pAb by Genscript; raised against synthetic peptide RVATAKNRMC) and with the corresponding secondary antibody (anti-rabbit IgG-peroxidase conjugate (Sigma) and anti-mouse IgG-alkaline phosphatase conjugate (Sigma)).

Techniques: Bacteria, Incubation, Western Blot, Control, Comparison, Membrane

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/ijn.s54750

Figure Lengend Snippet: Figure 8 In vivo blood–brain barrier (BBB) penetration. Notes: Representative fluorescence photomicrographs of immunohistochemistry on brain sections, of the specified area (marked with orange box) using a 6× Histag antibody to detect the TIMP-1-Histag fusion protein. The nucleus was labeled with DAPI. The TIMP-1 NPs + Ps80 group shows labeling indicating BBB penetration of TIMP- 1 NPs + Ps80 as indicated by white arrows. Abbreviations: TIMP, tissue inhibitor of matrix metalloproteinases; DAPI, 4′,6-diamidino-2-phenylindole; NPs, nanoparticles; Ps80, polysorbate 80; PBS, phosphate-buffered saline.

Article Snippet: After 2 hours, slices were incubated overnight at 4°C with a rabbit polyclonal anti-6× Histag antibody (1:500) (Abcam).

Techniques: In Vivo, Fluorescence, Immunohistochemistry, Labeling, Saline

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: In vivo blood–brain barrier (BBB) penetration. Notes: Representative fluorescence photomicrographs of immunohistochemistry on brain sections, of the specified area (marked with orange box) using a 6× Histag antibody to detect the TIMP-1-Histag fusion protein. The nucleus was labeled with DAPI. The TIMP-1 NPs + Ps80 group shows labeling indicating BBB penetration of TIMP-1 NPs + Ps80 as indicated by white arrows. Abbreviations: TIMP, tissue inhibitor of matrix metalloproteinases; DAPI, 4′,6-diamidino-2-phenylindole; NPs, nanoparticles; Ps80, polysorbate 80; PBS, phosphate-buffered saline.

Article Snippet: For detection, the membrane was incubated overnight in 1:200 dilution of mouse anti-TIMP-1 antibody (AF980; R&D Systems, Minneapolis, MN, USA), and rabbit polyclonal 6× Histag antibody (ab9108; Abcam, Cambridge, UK) in TBST at 4°C.

Techniques: In Vivo, Fluorescence, Immunohistochemistry, Labeling, Saline