Journal: Scientific Reports

Article Title: Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains

doi: 10.1038/s41598-017-19003-4

Figure Lengend Snippet: Asn209 in the constant region of mouse IgM heavy chain is crucial for extracellular cleavage of the antibody. ( a ) The scheme presents residues from the loop containing Asn209 that were subjected to alanine screening. Glycine residues at P3 and P4 were not mutated. ( b – e ) Stability of mutated IgMs in the presence of serum. μ’ chain was detected using western blotting. The samples were probed with anti-mouse-IgMκ antibody. ( b ) Stability of muteins generated using alanine screening. ( c ) Stability of Asn209Asp and Asp212Ser (N-glycosylated Asn209) muteins. Asn209Asp mutein was incubated at neutral or acidic pH. Despite many efforts we were not able to produce Arg210Pro mutein. Asn209 glycosylation was confirmed by a band shift visible on membrane stained with Coomassie Brilliant Blue (CBB). The full-length membrane is presented in Supplementary Figure . Contrast of the Coomassie-stained membrane was enhanced equally across the entire image. All bands remained visible after the digital processing. ( d ) Stability of IgMs with P1 position mutated into other 19 amino acids. Analyzed samples were derived from the same experiment but resolved in two different gels because of the limited number of wells. The figure presents images of two different blots processed in parallel. ( e ) Stability of chimeric mouse/human IgM. ( f ) Functional affinity of mutated IgMs analyzed by ELISA on immobilized human erythrocytes bearing a cognate antigen . The results are representatives of three ( b and e ) or two ( c , d , f ) independent experiments. Bands corresponding to HC in blots presented in panels c and d are slightly overexposed in order to make μ’ signal more visible. Serial exposures of the overexposed blots are provided in Supplementary Figure .

Article Snippet: The samples were analyzed by western blotting using HRP-labeled goat anti-mouse IgMκ antibody (AbD Serotec, Cat# 5276–2504).

Techniques: Western Blot, Generated, Incubation, Electrophoretic Mobility Shift Assay, Staining, Derivative Assay, Functional Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains

doi: 10.1038/s41598-017-19003-4

Figure Lengend Snippet: Legumain does not cleave mouse IgM. ( a ) IgM cleavage in serum treated with alkylating agents. Alkylating agents, such as iodoacetamide or NEM, are potent inhibitors of legumain – a cysteine protease specific to Asn at P1 site. Activity of legumain present in FBS was tested using fluorogenic substrate Z-Ala-Ala-Asn-AMC. Samples were analyzed using western blotting with anti-mouse IgMκ antibody. The full-length blot is presented in Supplementary Figure . ( b ) Recombinant legumain did not cleave IgMs. The gel was stained with Coomassie BB. ( a and b ) Representatives of two independent experiments are shown.

Article Snippet: The samples were analyzed by western blotting using HRP-labeled goat anti-mouse IgMκ antibody (AbD Serotec, Cat# 5276–2504).

Techniques: Activity Assay, Western Blot, Recombinant, Staining

Journal: Scientific Reports

Article Title: Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains

doi: 10.1038/s41598-017-19003-4

Figure Lengend Snippet: Factors affecting IgM cleavage. Samples were analyzed using western blotting with anti-mouse IgMκ antibody. ( a ) Serum-induced IgM cleavage in the presence of protease inhibitors. Presented result is an example of many experiments, in which also other inhibitors, all listed in Methods section, were used. None of the single inhibitors prevented IgM trimming. ( b ) Serum-induced IgM cleavage at different pH. Standard sample – medium without additional buffer. ( c and d ) IgM cleavage induced by serum-free medium collected from Hep G2 cell culture. The medium was concentrated by ultrafiltration with 3 kDa cut-off. Cibacron Blue resin removes a factor cleaving IgM from the medium ( d ). The image in panel c combines data extracted from one blot presented in full in Supplementary Figure . ( e ) IgM cleavage induced by various proteins. IgM was incubated with a hundredfold excess of the indicated proteins. Samples were analyzed in duplicates or triplicates. ( f ) IgM stability in the presence of protamine. Representatives of ten ( a ), two ( b , e , f ), or four ( c , d ) independent experiments are shown.

Article Snippet: The samples were analyzed by western blotting using HRP-labeled goat anti-mouse IgMκ antibody (AbD Serotec, Cat# 5276–2504).

Techniques: Western Blot, Cell Culture, Incubation