Caps buffer

Bk-343-HOPO and Bk-DTPA XAS samples were previously prepared as part of samples containing both ²⁴⁹Bk and ²⁴⁹Cf (Deblonde, Kelley et al., 2018 ▸ ; Kelley et al., 2018 ▸ ), and details are included here for completeness. For both 343-HOPO and DTPA, samples containing both ²⁴⁹Bk(III) and ²⁴⁹Cf(III) (ratio Cf/Bk = 1.9) were prepared using a ²⁴⁹BkCl₃ stock solution in 0.1 M HCl that had decayed for 510 days to allow for in-growth of the ²⁴⁹Cf daughter. The Bk/Cf samples were prepared using a metal:ligand ratio of 1:1.3 to ensure complete metal–ion complexation and assembled from aliquots of the metal stock solution with either a 343-HOPO stock solution in water or a DTPA stock solution at pH 4. The metal–ligand complexes were diluted with CAPS buffer (Sigma–Aldrich, BioUltra, >99%) to volumes of ∼65 µl to yield final concentrations of 0.11 mM for ²⁴⁹Bk and 0.20 mM for ²⁴⁹Cf. The pH values of the samples were buffered to 7–8 and each solution was loaded into separate, triply contained, aluminium holders (analogous to Pu) within ten days of synchrotron measurement.

The N-termini of the heavy chain, light chain, and Protac-digested heavy chain were analyzed by Edman degradation [37 ]. Purified proteins were subjected to reducing 12% SDS-PAGE and electrotransfered overnight at 30 V in CAPS buffer (Sigma-Aldrich, Oakville, ON) onto Immobilon-P^SQ PVDF membrane (Millipore, Billerica, MA). The membrane was incubated for 10 mins in 0.2% (w/v) Ponceau S (Sigma-Aldrich, Oakville, ON) in 1% (v/v) acetic acid and washed three times in Milli-Q water. It was then stored in a moist chamber and submitted for sequencing (The Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, ON). Stained protein bands were excised and analyzed by the Procise Protein Sequencing System (Applied Biosystems, Foster City, CA) as previously described [38 ].

Pectate lyase from Aspergillus (Megazyme, 180 U/mg, Cat # E-PCLYAN2) at 4.7U/mL (8uL of stock solution in 5mL of 50 mM CAPS buffer, Sigma-Aldrich A4926; pH 10)¹⁰ (link) and BAPTA calcium chelation at 2mM (Sigma Aldrich – Cat # A4926) treatments were performed on cell wall peels generated as described above. Treatments were carried out for 3 hours and 10 min, respectively, on the peels.
To screen for the effectivity of the treatments prior to vitrification, staining of non-treated, BAPTA- or PL- treated onion peels by a homogalacturonan-specific probe, Chitosan OligoSaccharide coupled with Alexa-488 (COS488) (Figure S6B) was performed based on a protocol provided by Jozef Mravec (personal communication): 1:1000 dilution from the mother solution kindly provided by Jozef Mravec (kept at -20C wrapped in foil) in 50 mM MES buffer pH 5.8 for 15 min. Peels were then washed with DI water 3 consecutive times before being mounted between a slide and coverslip and then screened by confocal laser scanning microscopy.⁵³ (link)