CaMKII specific activity after purification.

Radiolabeled ATP assay shows a linear response for at least 90 seconds. Specific activity is 5 μmol/min/mg. Error bars represent n = 3. (TIF)</p

Effect of pH on CaMKII purification with cation-exchange chromatography.

Initial separation of CaMKII from clarified lysate at (A) pH 7.2, (B) pH 7.5, and (C) pH 8.0. SDS-PAGE followed by Coomassie stain directly corresponds to the Western blots in Fig 3. (TIF)</p

Liquid chromatography mass spectrometry detection of CaMKIIα isoform B phosphorylation at the nuclear localization sequence (Ser 332/333/334/335).

Liquid chromatography mass spectrometry detection of CaMKIIα isoform B phosphorylation at the nuclear localization sequence (Ser 332/333/334/335).</p

Representative SDS-PAGE Coomassie gel and 6G9 Western blot of the two-step Mono S / CaM-Sepharose purification.

(A). SDS-PAGE followed by Coomassie stain, (B) Western blots stained with 6G9 anti-CaMKII primary antibody and detected with IRDye680RD secondary antibody. (TIF)</p

Mono S chromatograms of CaMKII crude separation at three different working pH values.

Mono S chromatograms of CaMKII crude separation at three different working pH values.</p

Side-by-side comparison of CaMKII initial separation by either phosphocellulose or Mono S cation exchange chromatography, normalized by a second CaM-Sepharose affinity chromatography step.

SDS-PAGE followed by Coomassie stain (A and C) or Western blots stained with 6G9 anti-CaMKII primary antibody and detected with IRDye680RD secondary antibody (B and D). (A and B) use phosphocellulose for cation-exchange chromatography, and (C and D) use Mono S for cation-exchange chromatography. Lanes were loaded with equal volumes. Lane representation: IN–cation exchange chromatography combined elution fractions; FT–calmodulin-Sepharose flow through, WASH 1–3 –post-incubation wash steps, ELUTION 1–2 –elution column volumes.</p

Characterization of two-stage purified CaMKII obtained from BEVS.

(A) Native PAGE followed by Coomassie stain. (B) Superose 6 size-exclusion chromatogram. (C) Representative negative stain TEM micrograph of CaMKII oligomers. Scale bar 20 nm.</p

Western blot analysis of CaMKII expression in clarified lysate as a function of time and expression system.

(A) E. coli expression at various timepoints detected with primary antibody anti-CaMKII 6G9 followed by IRDye 680RD. (B) E. coli expression (18 h) detected with primary antibody anti-phospho-CaMKII 22B1 followed by IRDye 680RD reveals no presence of Thr286 phosphorylation. (C) Tni expression at various timepoints detected with primary antibody anti-CaMKII 6G9 followed by IRDye 680RD. (D) Tni expression (72 h) detected with primary antibody anti-phospho-CaMKII 22B1 followed by IRDye 680RD reveals no presence of Thr286 phosphorylation.</p

Schematic detailing the comparison of the side-by-side two-step purification scheme.

The difference in column volume sizes between Mono S and phosphocellulose are normalized by the CaM-Sepharose step, thereby making protein concentration measurement between the two methods equivalent. Legend: CV–column volume.</p

Analysis of baculovirus/insect expression parameters on the expression of CaMKII.

All figures are Western blots stained with primary antibody anti-CaMKII 6G9 followed by IRDye 680RD. (A) Effect of seeding density of Tni cells before infection. (B) Effect of virus concentration at multiplicity of infections of 0.32, 3.2, and 16. (C) Effect of 5% BPA added 24 h post-infection (-BPA is no BPA added).</p