sds page concentrated gel buffer  (Bio-Rad)

Journal: Toxins

Article Title: Cholera Toxin-Mediated Targeting of Botulinum Neurotoxin Activity to Pain-Associated Sensory Neurons

doi: 10.3390/toxins18040174

Figure Lengend Snippet: Production of ChoBot: ( a ) Schematic showing self-assembly of the AB5-linker, a CTB pentamer assembled around CTA2 fused to Linker 1, CTA2-cholera toxin A2 subunit, CTB-cholera toxin B subunits; ( b ) coomassie-stained SDS-PAGE gel showing that mixing the linker 1-AB5 and the light chain/translocation domain (LcTd) of BoNT/A attached to Linker 2 (LcTd-Linker 2) in the presence of Linker 3 (not visible due to low MW) results in formation of LcTd-Linkers-CTA2 (arrowhead). CTB5 is denoted as a pentamer of approx. 50 kDa. Note, the CTB5 and CTA2 association is not SDS-resistant and therefore only the linking of LcTd to CTA2 is visible as a slight shift in MW of LcTd-Linker 2. Boiling for 2 min in the SDS-PAGE sample buffer results in dissociation of CTB5 pentamer into monomeric B subunits. Molecular weight standards are denoted as MW; ( c ) schematic of ChoBot modelled using known X-ray structures of the BoNT/A, the SDS-resistant SNARE linking complex and cholera toxin (PDBs 3BTA, 1SFC and 1XTC, respectively).

Article Snippet: Cell culture medium was then removed and cells were lysed in 45 μL of SDS-PAGE running buffer (56 mM sodium dodecyl sulphate, 0.05 M Tris-HCl, pH 6.8, 1.6 mM EDTA, 6.25% glycerol, 0.0001% bromophenol blue, 10 mM MgCl2, 26 U/mL benzonase) by shaking at 900 rpm for 10 min. Lysed cells were then boiled at 95 °C for 3 min and then run on 12% Novex SDS–PAGE gels (Invitrogen) for 3 h at 4 °C to increase separation between cleaved and intact SNAP-25.

Techniques: Staining, SDS Page, Translocation Assay, Molecular Weight