tbe denaturing polyacrylamide gel  (Thermo Fisher)

Journal: bioRxiv

Article Title: Component A2 is a redox-sensitive archaeal ATPase activated by methyl-coenzyme M reductase

doi: 10.64898/2026.03.18.712670

Figure Lengend Snippet: (a) Genomic arrangement of the MCR activation operon in M. acetivorans with scale bar depicting 1 kilobase pair. (b) Structure of the putative MCR activation complex (PDB: 9H1L) with MCR in orange, component A2 (with relevant domains highlighted) in blue, and the other methanogenesis marker proteins in gray. NBD refers to N ucleotide B inding D omain and ZBM refers to Z inc B inding M otif. (c) Log 2 transformed FPKM ( F ragments P er K ilobase of transcript per M illion mapped reads) of the MCR activation operon and the MCR operon in M. acetivorans grown in high-salt (HS) minimal medium supplemented with trimethylamine (TMA) at 37 °C. (d) Genotype of an M. acetivorans strain expressing a second copy of component A2 in trans under the control of a tetracycline inducible promotor. (e) Anti-FLAG immunoblot showing the inducible production of component A2 upon the addition of 100 µg/mL tetracycline (tet) to the growth medium in crude and soluble cell lysates with 13.9 µg total protein loaded into each lane. (f) Anti-FLAG immunoblot of aerobic affinity-purification of A2 with a Streptactin resin. The lanes represent the following: (1) ladder, (2) cell lysate, (3) flow-through, (4) first wash, (5) second wash, (6) first elution, (7) second elution, and (8) third elution. (g) SDS-PAGE gel showing anaerobic purification of full-length TAP-tagged component A2 (64 kDa with tag). (h) Anaerobic ATPase assay of component A2 alone (blue), MCR alone (purple), and component A2 combined with MCR (orange). Each reaction contained 500 µg/mL of each protein indicated with 200 µM ATP, 10 mM MgCl 2 , 20 mM HEPES, 300 mM NaCl, and 1% glycerol. Reactions were incubated at 37 °C. Inorganic phosphate production was measured at 0, 15, 30, and 60 minutes using the malachite green reagent. (I) Aerobic ATPase assay of component A2 alone (blue), MCR alone (purple), and component A2 combined with MCR (orange). Proteins were purified anaerobically then removed from the anaerobic chamber and reactions were set up on the bench top. The same assay conditions as (c) were used, but time points were taken at 0, 30, and 60 minutes. Error bars represent the standard deviation of three technical replicates for each reaction.

Article Snippet: Samples were then loaded into 12% precast Tris-Glycine denaturing SDS-PAGE gels (Mini-PROTEAN TGX, Bio-Rad, Hercules, CA, USA).

Techniques: Activation Assay, Marker, Transformation Assay, Expressing, Control, Western Blot, Affinity Purification, SDS Page, Purification, ATPase Assay, Incubation, Standard Deviation