HXK1 | Hexokinase 1 (Chlamydomonas)
AS16 4083 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Chlamydomonas reinhardtii, Eudorina elegans, Gonium pectorale
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1 : 1000 (WB)
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Total protein of Chlamydomonas reinhardtii strain 21gr (1), strain cw15 (2), strain M10 (3) was extracted using 1x Laemmli buffer + DTT + protease and phosphatase inhibitors without bromophenol blue heated at 95°C for ten minutes then vortexed with zirconia beads at max speed for ten minutes. Lysate was spun at 4°C for ten minutes at max speed in a tabletop centrifuge. Proteins were precipitated out of supernatant in 80% acetone at -20°C for 20 min, spun at 4°C 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 1 µg chlorophyll/uL. Protein equivalent to1 of chlorophyll was loaded on a 4-20% Bis-Tris gel and blotted to PVDF using wet transfer methods. Blot was blocked with 10% 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/4°C. 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 1:50 000 (AS09 602) in 5% milk in TBS-T for 1hr/RT with agitation. The blot was washed as above and developed withchemiluminescent detection reagent and imaged on a Licor C-Digit blot scanner. Exposure time was 12 min.
Courtesy of Jessica Rakijas, Kansas State University, USA
HXK1 (Hexokinase 1) is an enzyme of plant glucose-signaling network which functions as a glucose sensor. Alternative name: Protein GLUCOSE INSENSITIVE 2.
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