Kelch repeat protein (Chlamydomonas)
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Recombinant Cr-Klech, UniProt: A8J2W4, Locus name: g2987 (from Chlamydomonas Reinhardtii)
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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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