MA1-047

antibody from Invitrogen Antibodies

Targeting: CAMK2A CAMKA, CaMKIINalpha, CaMKIIα, KIAA0968

Provider product page for MA1-047

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Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [80]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [4]
Other assay [10]

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Product number: MA1-047 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-CaMKII alpha (Thr286) Monoclonal Antibody (22B1)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA1-047 detects phosphorylated CaM kinase II from human, rat and mouse tissues/cells. This antibody has been shown to detect the phosphorylated alpha subunit CaM kinase II only. MA1-047 does not detect the non-phosphorylated alpha subunit CaM kinase II nor with the ~60 kDa beta subunit in either phosphorylation state. MA1-047 has been successfully used in Western blot, immunofluorescence, immunoprecipitation, immunocytochemistry, immunohistochemistry, and ELISA procedures. By Western blot, this antibody detects an ~50 kDa band representing the alpha subunit of CaM kinase II only after phosphorylation. Immunofluorescence staining of CaM kinase II in rat/mouse hippocampal cells with MA1-047 densely labels cell somas and neuropil areas, while nuclei are only lightly stained. The MA1-047 immunizing peptide is a synthetic peptide corresponding to residues M(281) H R Q E T(p) V D C L K K F N(294) of rat CaM Kinase II protein with a phosphorylated threonine at position 286.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 22B1
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

CAMK2A protein structure - MA1-047 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-CaM Kinase II pThr286 using Anti-Phospho-CaM Kinase II pThr286 Monoclonal Antibody (22B1) (Product # MA1-047) shows staining in C6 Cells. Phospho-CaM Kinase II pThr286 staining (green) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Phospho-CaM Kinase II pThr286 (Product # MA1-047) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-CaM Kinase II pThr286 using Anti-Phospho-CaM Kinase II pThr286 Monoclonal Antibody (22B1) (Product # MA1-047) shows staining in Hela Cells. Phospho-CaM Kinase II pThr286 staining (green) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Phospho-CaM Kinase II pThr286 (Product # MA1-047) at a dilution of 1:50 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-CaM Kinase II pThr286 using Anti-Phospho-CaM Kinase II pThr286 Monoclonal Antibody (22B1) (Product # MA1-047) shows staining in U251 Cells. Phospho-CaM Kinase II pThr286 staining (green) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Phospho-CaM Kinase II pThr286 (Product # MA1-047) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-CaM Kinase II pThr286 in Neuro-2a cells (left panel) or cells stimulated with Lamda-Phosphatase (Lambda-PPase) (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Phospho-CaM Kinase II pThr286 Monclonal Antibody (22B1) (Product # MA1-047) at a dilution of 1:50 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 11 Immunolocalization of CaMKIIdelta and phospho-CamKIIdelta at E11.5 in TgPCP4 and Ts1Cje. A,B : Immunohistochemistry of CaMKIIdelta (A) and phospho-CaMKIIdelta (B) on close sagittal sections of E11.5 wt embryos showing the localization of CaMKIIdelta in the nervous system at the pial surface of the neuroepithelium in the telencephalon (Tel), diencephalon (Di), mesencephalon (Mes), and rhombencephalon (Rh) and spinal cord (sp) and in the dorsal root ganglia (drg). Phospho-CaMKIIdelta showed a close pattern to CaMKIIdelta with less intense signals. The p-CaMKIIdelta signal is more intense at the level of the pial surface. C-T : Immunohistochemistry of phospho-CaMKIIdelta (p-CaMKIIdelta, green) and pcp4 (red) in (C-K) postmitotic neurons of the rhombencephalon (r6 level) and in (L-T) dorsal root ganglia in wt, TgPCP4, and Ts1Cje embryos corresponding to boxed area in B. Immunolocalization of p-CaMKIIdelta was enhanced in the rhombencephalon (D,E, arrows) and in the anterior roots of ganglia (M,N, arrows) in TgPCP4 and Ts1Cje compared with wt (C,L). U-X : Colocalization of CaMKIIdelta (green) and pcp4 (red) at E11.5 in regions corresponding to the boxed area in A. Note the more intense colabeling of CaMKIIdelta and pcp4 in the TgPCP4 postmitotic cells (pm) of the rhombencephalon (r5-r6 level) indicated by arrows (V) and dorsal root ganglia (DRG) (X). Scale bar = 500 mum in A,B; 50 mum in C-X.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Effects of vehicle and neuregulin treatment on calcium handling proteins not associated with the myofilament . A-F) Quantitation of proteins compared with respective total protein or GAPDH for each treatment group is shown. Values for hearts from non-treated (white bars), vehicle-treated (gray bars), and neuregulin-treated (black bars) animals are shown. Sample number is indicated within each bar. Significance was determined by 1-way ANOVA and Newman-Keuls multiple comparison test using GraphPad software. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Gender-associated changes in CaMKP and in CaMKII activation in cardiac subcelullar fractions 30 days after TAC. ( A ) Representative immunoblots of Oct1, GAPDH, CaMKP, CaMKII (phospho-Trh286 CamKII and total CaMKII) and HDAC4 (phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) and total HDAC) in cardiac subcellular fractions (nuclear (n) and cytoplasmic (c)) of mouse hearts of both genders 30 days after sham-operation (Sham) or thoracic aortic constriction (TAC). In histograms, blue bars indicate male mice (light blue bar: M Sham; dark blue bar: M TAC), and red bars indicate female mice (light red bar: F Sham; dark red bar: F TAC). ( B - C ) Quantification of CaMKP protein level in LV nuclear ( B ) or cytoplasmic ( C ) extracts reveals a marked gender-dependent up-regulation of CaMKP expression in LV samples after TAC. CaMKP protein levels are increased in both nuclear and cytoplasmic extracts in male mice only. ( D - E ) Phosphorylation status of CaMKII in LV nuclear ( D ) and cytoplasmic ( E ) extracts: phospho-Thr286 CaMKII (yellow bar); total CaMKII (green bar); pCaMKII/tCaMKII ratio (red bar). CaMKP and CaMKII protein levels are expressed as fold increases in TAC mice relative to their respective sham groups. N = 4-8 mice per group. *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 11 Immunolocalization of CaMKIIdelta and phospho-CamKIIdelta at E11.5 in TgPCP4 and Ts1Cje. A,B : Immunohistochemistry of CaMKIIdelta (A) and phospho-CaMKIIdelta (B) on close sagittal sections of E11.5 wt embryos showing the localization of CaMKIIdelta in the nervous system at the pial surface of the neuroepithelium in the telencephalon (Tel), diencephalon (Di), mesencephalon (Mes), and rhombencephalon (Rh) and spinal cord (sp) and in the dorsal root ganglia (drg). Phospho-CaMKIIdelta showed a close pattern to CaMKIIdelta with less intense signals. The p-CaMKIIdelta signal is more intense at the level of the pial surface. C-T : Immunohistochemistry of phospho-CaMKIIdelta (p-CaMKIIdelta, green) and pcp4 (red) in (C-K) postmitotic neurons of the rhombencephalon (r6 level) and in (L-T) dorsal root ganglia in wt, TgPCP4, and Ts1Cje embryos corresponding to boxed area in B. Immunolocalization of p-CaMKIIdelta was enhanced in the rhombencephalon (D,E, arrows) and in the anterior roots of ganglia (M,N, arrows) in TgPCP4 and Ts1Cje compared with wt (C,L). U-X : Colocalization of CaMKIIdelta (green) and pcp4 (red) at E11.5 in regions corresponding to the boxed area in A. Note the more intense colabeling of CaMKIIdelta and pcp4 in the TgPCP4 postmitotic cells (pm) of the rhombencephalon (r5-r6 level) indicated by arrows (V) and dorsal root ganglia (DRG) (X). Scale bar = 500 mum in A,B; 50 mum in C-X.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Effects of hyperaldosteronism and/or hypoestrogenism on activation of Ca 2+ -signaling pathway (Cn/ NFAT /Ca MKII ,) proteins in wild type ( WT ) or aldostereone synthase overexpressing ( AS ) mouse hearts. (A) shows representative immunoblots of activation of calcium-calmodulin-dependent kinase II (Ca MKII ): its oxidation (Ox-Ca MKII ) or its phosphorylation (phospho-Thr 286 ), and calcineurin (Cn), and nuclear factor of activated T-cells ( NFAT c4) and its phosphorylation (phospho-Ser 168/170 ), and its inhibitor, calcipressin ( MCIP ). (B-G) illustrated their respective quantifications, Ca MKII activity calculated as the ratio of Ox-Ca MKII to Ca MKII , and/or p-Ca MKII to Ca MKII . The ratio of p- NFATC 4 to NFATC 4 indirectly reflected Cn activity. Ca MKII and pan Cn and MCIP protein expression level normalized to that of GAPDH . The values expressed as mean +- SEM ( n = 5-10 in each group). * P < 0.05; ** P < 0.01; *** P < 0.001; # P < 0.05, and ## P < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Basal expression and phosphorylation of CaMKII is affected by PDE2 overexpression. ( A ) Representative immunoblots and ( B ) quantification of protein kinase expression normalized to GAPDH, CaMKII phosphorylation at Threonine 286 normalized to total CaMKIIdelta in lysates of isolated cardiomyocytes from PDE2 OE (orange) and WT (black) mice. (N = 6 animals/genotype. * p < 0.05, *** p < 0.001 vs. WT).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 GluN2A(N615S) containing NMDAR expression in vitro and in vivo. a The position of N615 in the membrane segment M2 is depicted together with the three channel-forming trans-membrane segments M1, 3, and 4. b In HEK293 cells, recombinantly expressed GluN1/GluN2A and GluN1/GluN2A(N615S) channels were activated by fast glutamate application (1 mM; in the continuous presence of the co-agonist glycine, 10 uM) at holding potentials from -100 to +40 mV in different extracellular Mg 2+ concentrations. NMDAR-mediated peak currents were normalized to those obtained at +40 mV. Data points represent mean +- SEM for n = 4-7 different HEK293 cells. Representative current traces evoked in 0 mM Mg 2+ at - 60 mV, with 20 and 600 ms applications, are shown below the IV plots and were used to determine the current kinetics (Supplementary Table 1 ). c Schematic view of the A to G replacement in exon 10 of the mouse Grin2a gene. d Reverse transcription PCR (RT-PCR)-sequence analyses of total brain mRNA show the A-to-G mutation in pos. 0 and two diagnostic silent mutations at pos. -11 and -14 in the pore loop encoding gene segment in Grin2a +/+ , Grin2a S/S , Grin2a +/S mice. In Grin2a +/S mice, the overlay of two different colored ""nucleotide"" peaks, at position 0, -11, and -14 indicate equimolar amounts of mRNA from the Grin2a + and the targeted Grin2a S alleles. e Immunoblots of forebrain protein lysates of 4-week-old mice (Supplementary Fig. 2 ) indicate no genotype-specific differences

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. L-type Ca 2&plus channels (LTCCs) at crests in rat failing myocytes are hyperphosphorylated by Ca 2&plus calmodulin-dependent protein kinase II (CaMKII). A , Mean phosphorylated CaMKII (Thr286) from whole-cell lysate in control and failing cardiac myocytes. A representative blot is shown above the graph. Results are normalized to &agr-tubulin ( B ) Mean p-CaMKII from whole tissue from control and failing human samples. A representative blot is shown above the graph. Results are normalized to GAPDH. C , Mean p-CaMKII from whole-cell lysate in failing cardiac myocytes with or without CaMKII inhibition. A representative blot is shown above the graph. Results are normalized to &agr-tubulin. D , LTCC open probability (P o ) in control and failing rat myocytes, under control conditions and after CaMKII inhibition with 5-&mgrmol&solL KN-93 or with 5-&mgrmol&solL autocamptide-2 related inhibitor peptide (AIP; control T-tubule &lsqbTT&rsqb n=19; control TT&plusKN-93 n=5; failing TT n=11; failing TT&plusKN-93 n=4; control crest n=4; failing crest n=13; failing crest&plusKN-93 n=9; failing crest&plusAIP n=3. &ast P