Journal: Frontiers in Pharmacology

Article Title: Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies

doi: 10.3389/fphar.2020.594755

Figure Lengend Snippet: Detection of modification of HSA by CLV-tetraethylenglycol-Biotin (CLV-TEG-B). Human serum albumin (HSA) was incubated in the presence of CLV-TEG-B for 16 h at 37°C. HSA:CLV-TEG-B molar ratios are shown in figure (A) Aliquots of the haptenation reactions containing 2 µg of protein were analyzed by SDS-PAGE and CLV-TEG-B modification was detected by blot with streptavidin-HRP and ECL detection (B) 0.15 µg aliquots of HSA-CLV-TEG-B (left) and 0.006–0.377 pmol aliquots of BSA-B standards (right) were analyzed by SDS-PAGE followed by blot with streptavidin-HRP and ECL detection.

Article Snippet: All conjugates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by modification detection with streptavidin-horseradish peroxidase (HRP) and ECL , as described below.

Techniques: Modification, Incubation, SDS Page

Journal: Frontiers in Pharmacology

Article Title: Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies

doi: 10.3389/fphar.2020.594755

Figure Lengend Snippet: Detection of modification of serum proteins by clavulanic acid (CLV)-tetraethylenglycol-Biotin (CLV-TEG-B) and identification of targets. Human sera were incubated in the presence of CLV-TEG-B for 16 h at 37°C. (A) 4 µg aliquots of resulting adducts were analyzed by SDS-PAGE and CLV-TEG-B modification was detected by blot with streptavidin-HRP and ECL detection. Lower panel shows the Coomassie staining for total proteins visualization. (B) 50 µg aliquots of resulting adducts were subjected to 2D-eletrophoresis on duplicate gels, after which, one of the gels was used for detection of modified proteins by transfer to membrane and biotin detection with avidin-HRP and the other one was used for protein staining with Coomassie. Matched spots were excised from the gel and used for tryptic digestion and peptide fingerprint analysis. The blot in the middle panel is deliberately overexposed in order to show the signals corresponding to modified immunoglobulin chains. The red arrow point towards a spot that could be proposed as transferrin, on the bases of its molecular weight and isoelectric point.

Article Snippet: All conjugates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by modification detection with streptavidin-horseradish peroxidase (HRP) and ECL , as described below.

Techniques: Modification, Incubation, SDS Page, Staining, Avidin-Biotin Assay, Molecular Weight

Journal: Frontiers in Pharmacology

Article Title: Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies

doi: 10.3389/fphar.2020.594755

Figure Lengend Snippet: Competition assay between clavulanic acid (CLV) and CLV-tetraethylenglycol-Biotin (CLV-TEG-B). Human serum albumin (HSA) was preincubated with increasing concentrations of CLV (80, 800 and 8,000 μM) for 16 h at 37°C and then with a fixed concentration of CLV-TEG-B (80 μM) for 2 h at 37°C. Resulting adducts were analyzed by SDS-PAGE and CLV-TEG-B modification was detected by blot with streptavidin-HRP and ECL detection (top panel) . Total protein was detected by Coomassie staining (bottom panel) .

Article Snippet: All conjugates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by modification detection with streptavidin-horseradish peroxidase (HRP) and ECL , as described below.

Techniques: Competitive Binding Assay, Concentration Assay, SDS Page, Modification, Staining