Kelch repeat protein (Chlamydomonas)
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Recombinant Cr-Klech, UniProt: A8J2W4, Locus name: g2987 (from Chlamydomonas Reinhardtii)
for reconstitution add 50 µl of sterile water.
Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
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no confirmed exceptions from predicted reactivity known in the moment
Total protein from Chlamydomonas reinhardtii (1), Gonium Pectorale (2), Eudorina elegans (3) was extracted using 1x Laemmli buffer + DTT + protease and phosphatase inhibitors without bromophenol blue heated at 95 oC for ten minutes then vortexed with zirconia beads at max speed for ten minutes. Lysate was spun at 4 oC for ten minutes at max speed in a tabletop centrifuge. Proteins were precipitated out of supernatant in 80% acetone at -20 oC for 20 min, spun at 4 oC at max speed for 10 min. Pellet was washed 1x with 80% acetone and resuspended in 1x LDS sample buffer + DTT + inhibitors at a concentration of 100 mg chlorophyll/uL. Protein equivalent to 1 ug of chlorophyll was loaded on a 4-20% Bis-Tris gel and blotted to nitrocellulose using wet transfer methods. Blot was blocked with 5% milk in TBS-T for 1hr/RT. Blot was incubated in the primary antibody at a dilution of 1:1000 in 5% milk in TBS-T ON/4oC. The antibody solution was decanted and the blot was rinsed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary diluted to 1:25,000 in 5% milk in TBS-T for 1hr/RT with agitation. The blot was washed as above and developed with chemiluminescent detection reagent and imaged on a Licor C-Digit blot scanner. Exposure time was 12 min.
Courtesy of Jessica Rakijas, Kansas State University, USA
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