Journal: bioRxiv

Article Title: Biochemical characterisation of human transglutaminase 4

doi: 10.1101/2021.09.01.458359

Figure Lengend Snippet: (A) BPA incorporation via hTG4 transamidase activity into AD-293 cell extract examined by Western blot, using Streptavidin/HRP for detection. The bottom part of the Western blot picture shows the presence of hTG4 in the sample, detected using anti-polyHis-tag/HRP. The vertical lines on the blot are labelling the place of the membrane cuttings. Representative picture from 2 independent experiments. The reaction conditions are summarised in the picture. BPA incorporation into AD-293 cytoplasmic (Cp) and nuclear (N) fractions directly after the enzyme reaction (B) and after the separation of the BPA containing substrates by NeutrAvidine Agarose (C). Western blots using Streptavidin/HRP for detection.

Article Snippet: The following antibodies were used in the study: Monoclonal Transglutaminase-4 antibody (1C6) (Covalab) (1/2500), Polyclonal antibody to human epidermal transglutaminase (TG3) (Zedira) (1/2000), Monoclonal anti-polyHis/HRP antibody (Sigma) (1/20000), Streptavidin/HRP (1/2500) (BioLegend), Goat-anti-mouse IgG (Advansta) (1/10000) and Goat-anti-rabbit IgG (H+L), HRP conjugate (Advansta) (1/10000).

Techniques: Activity Assay, Western Blot

Journal:

Article Title: Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of Escherichia coli

doi: 10.1128/IAI.69.12.7293-7303.2001

Figure Lengend Snippet: MBP-EibA fusions. Broth cultures were grown and induced with IPTG, and whole-cell were extracts prepared as described in Materials and Methods. Equivalent amounts of cell extracts were fractionated by SDS-PAGE (8% acrylamide) and blotted to PVDF. The blot was incubated first with human IgG Fc-HRP (A) and second with rabbit anti-MBP developed with donkey anti-rabbit Ig (B) (see Materials and Methods). Lanes: 1, pMal-c2X; 2, EibA (amino acids [aa] 254 to 392), pDC2240; 3, EibA (aa 254 to 344), pDC2283; 4, EibA (aa 300 to 392), pDC2252; 5, EibA (aa 254 to 298), pCS7299; 6, EibF (aa 318 to 459), pCS7280.

Article Snippet: Purified IgG Fc conjugated with HRP (IgG Fc-HRP) (Rockland) was used at a concentration of 20 ng of antibody per ml; purified whole human serum IgA conjugated with HRP (IgA-HRP) (Jackson ImmunoResearch Laboratories) was used at 50 ng per ml.

Techniques: SDS Page, Incubation

Journal:

Article Title: Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of Escherichia coli

doi: 10.1128/IAI.69.12.7293-7303.2001

Figure Lengend Snippet: MBP-EibF fusions. Blots were prepared as described in the legend to Fig. ​Fig.44 except that the amount of extract loaded in lane 7 was four times the volume loaded in other lanes. The blots were incubated sequentially, first with IgA-HRP (A), second with IgG Fc-HRP (B), and finally with rabbit anti-MBP developed with donkey anti-rabbit Ig-HRP (C) (see Materials and Methods). Lanes: 1, EibF (aa 97 to 459), pCS7269; 2, EibF (aa 97 to 399), pCS7285; 3, EibF (aa 97 to 353), pCS7296; 4, EibF (aa 318 to 459), pCS7280; 5, EibF (aa 181 to 280), pCS7286; 6, EibF (aa 400 to 459), pCS7284; 7, EibF (aa 181 to 280), pCS7286.

Article Snippet: Purified IgG Fc conjugated with HRP (IgG Fc-HRP) (Rockland) was used at a concentration of 20 ng of antibody per ml; purified whole human serum IgA conjugated with HRP (IgA-HRP) (Jackson ImmunoResearch Laboratories) was used at 50 ng per ml.

Techniques: Incubation

Journal:

Article Title: Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of Escherichia coli

doi: 10.1128/IAI.69.12.7293-7303.2001

Figure Lengend Snippet: Ig binding of ECOR strains. Whole-cell extracts from 24-h broth cultures were fractionated by SDS-PAGE (8% acrylamide) and blotted to polyvinylidene difluoride (PVDF). The blot was incubated sequentially with human IgG Fc-HRP (B) and IgA-HRP (A). Lanes: 1, ECOR-2; 2, ECOR-5; 3, ECOR-9; 4, ECOR-12; 5, ECOR-43; 6, ECOR-72. Each lane contained approximately 30 μg of protein.

Article Snippet: Purified IgG Fc conjugated with HRP (IgG Fc-HRP) (Rockland) was used at a concentration of 20 ng of antibody per ml; purified whole human serum IgA conjugated with HRP (IgA-HRP) (Jackson ImmunoResearch Laboratories) was used at 50 ng per ml.

Techniques: Binding Assay, SDS Page, Incubation

Journal:

Article Title: Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of Escherichia coli

doi: 10.1128/IAI.69.12.7293-7303.2001

Figure Lengend Snippet: Ig binding conferred by cloned eib genes. Extracts were prepared and fractionated on duplicate gels by SDS-PAGE (8% acrylamide) as shown in Fig. ​Fig.1.1. One gel was blotted to PVDF and the other was stained with Coomassie brilliant blue (C). The blot was incubated sequentially with IgG Fc-HRP (B) and IgA (A). Lanes: 1, positive control ECOR-2; 2, negative control pOK12; 3, eibA(pCS6379); 4, eibC(pCS6431); 5, eibD(pCS6364); 6, eibE(pCS6432); 7, eibF(pCS7216). Each lane contained approximately 10 μg of protein. E. coli DH5α was used as the background strain for all constructs (lanes 2 to 7).

Article Snippet: Purified IgG Fc conjugated with HRP (IgG Fc-HRP) (Rockland) was used at a concentration of 20 ng of antibody per ml; purified whole human serum IgA conjugated with HRP (IgA-HRP) (Jackson ImmunoResearch Laboratories) was used at 50 ng per ml.

Techniques: Binding Assay, Clone Assay, SDS Page, Staining, Incubation, Positive Control, Negative Control, Construct

Journal:

Article Title: Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of Escherichia coli

doi: 10.1128/IAI.69.12.7293-7303.2001

Figure Lengend Snippet: Trypsin treatment of cells expressing EibA or EibF. Whole cells containing cloned eibA (lanes 1 to 4) or eibF (lanes 5 to 8) were treated with trypsin at concentrations of 5 (lanes 2 and 6), 50 (lanes 3 and 7), or 500 (lanes 4 and 8) μg per ml (see Materials and Methods). Whole-cell extracts were prepared and fractionated on duplicate gels by SDS-PAGE (8% polyacrylamide) as described in the legend to Fig. ​Fig.2.2. One gel was blotted to PVDF and then incubated sequentially with IgG Fc-HRP (A) and IgA-HRP (B). The second gel was stained with Coomassie brilliant blue (C). Arrows, EibA (lane 1) and EibF (lane 5) when trypsin was not added.

Article Snippet: Purified IgG Fc conjugated with HRP (IgG Fc-HRP) (Rockland) was used at a concentration of 20 ng of antibody per ml; purified whole human serum IgA conjugated with HRP (IgA-HRP) (Jackson ImmunoResearch Laboratories) was used at 50 ng per ml.

Techniques: Expressing, Clone Assay, SDS Page, Incubation, Staining