13-7300

antibody from Invitrogen Antibodies

Targeting: CAMK2A CAMKA, CaMKIINalpha, CaMKIIα, KIAA0968

Provider product page for 13-7300

Western blot

Other assay

Antibody data

Antibody Data
Antigen structure
References [21]
Comments [0]
Validations
Western blot [4]
Other assay [17]

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Product number: 13-7300 - Provider product page
Provider: Invitrogen Antibodies
Product name: CaMKII alpha Monoclonal Antibody (Cba-2)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: This antibody is specific for alpha subunit of CaM Kinase II. No significant reactivity was seen with the B, B1, yB, dA, dB, and dC isoforms. The epitope is located in the regulatory domain.
Antibody clone number: Cba-2
Concentration: 0.5 mg/mL

CAMK2A protein structure - 13-7300 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on tissue extracts (30 µg lysate) of Rat Brain (Lane 1), and Mouse Brain (Lane 2). The blots were probed with Anti-CaM Kinase II alpha Mouse Monoclonal Antibody (Product # 13-7300, 0.5-1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjµgate (Product # 62-6520, 1:4000 dilution). A ~ 54 kDa band corresponding to CaM Kinase II alpha was observed across tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot using CaM Kinase II Alpha Monoclonal Antibody, Mouse: Western blot analysis of mouse brain lysates using CaM Kinase II Alpha Monoclonal Antibody, Mouse (lane 1, Product # 13-7300) and b-CAM Kinase II Monoclonal Antibody, Mouse (lane 2, Product # 13-9800).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on tissue extracts (30 µg lysate) of Rat Brain (Lane 1), and Mouse Brain (Lane 2). The blots were probed with Anti-CaM Kinase II alpha Mouse Monoclonal Antibody (Product # 13-7300, 0.5-1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjµgate (Product # 62-6520, 1:4000 dilution). A ~ 54 kDa band corresponding to CaM Kinase II alpha was observed across tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-CaMKII alpha Monoclonal Antibody (Cba-2) (Product # 13-7300) and a band at ~50 kDa corresponding to CaMKII alpha was observed in Mouse Brain in comparison to other tissues which are reported to be negative. Tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Mouse Skeletal Muscle (Lane 2) and Mouse Heart (Lane 3) were electrophoresed using Novex® NuPAGE® 4-12% % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1µg/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Relative content of phosphorylated Ca 2+ /CaM Kinase II, Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 in hippocampal slices incubated with Melatonin. Hippocampal slices were incubated for 3 h with either the vehicle (VEH) or 100 nM melatonin (MEL). CaMKII and Thr286-phospho-CaMKII (P-CaMKII), PKC and phospho-PKC (P-PKC) as well as ERK1/2 and phospho-ERK1/2 (P-ERK1/2) were determined by Western blot. Upper panels show immunodetection of GAPDH as load control. Representative fluorograms of total and phosphorylated enzymes revealed by ECL are shown below. Histograms correspond to densitometric analysis of bands depicted immediately above. Results are expressed as the mean +- SEM of four densitometric scannings obtained from two independent experiments. Asterisks indicate significant differences ( p < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Calmodulin content in hippocampal slices treated with Melatonin. Hippocampal slices were incubated during 6 h with either the vehicle (VEH) or melatonin (MEL). Calmodulin (CaM) in the homogenates ( A ); and in the soluble (SOL) and cytoskeletal fractions (CSK) separated by centrifugation ( B ); was determined by Western blot. Upper panels show Carbonic Anhydrase (CA) used as external load control and stained with Coomassie blue. Representative fluorograms of CaM are shown immediately below. First lane of both gel and fluorogram from panel A was loaded with pure CA (5 ug) and CaM (1 ug), respectively. CaM was recognized with a specific CaM antibody and ECL. Histograms correspond to densitometric analysis of the bands shown in the upper panels. Results are the mean +- SEM of four densitometric scannings obtained from two independent experiments. Asterisk indicates significant differences ( p < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Verification of phosphoregulated Camk2a, ERK1, and ERK2 by immunoblotting (A) Western blot (WB) images for pCamk2a (pThr 286 ) and total Camk2a. Analyses were performed using three independent biological replicates of Bic-treated, TTX-treated, and untreated (control) neurons for 5 min (top) or 24 h (bottom). (B) Bar graphs show quantification of pCamk2a detected by WB in comparison to the phosphopeptide's signals quantified via LC-MS/MS (MS) after 5 min (left) or 24 h (right) Bic or TTX stimulation. Protein signals of the blot were quantified based on the intensity ratio of the phosphorylated protein over total protein normalizing treated conditions to the untreated control. Log 2 -scaled MS intensity of the phosphorylated peptide is depicted as the fold change of treatment over control as well (see Method details ). Error bars represent the standard deviation between biological replicates (WB, n = 3; MS, n = 4). For statistical analysis of the WB data, a two-sided t test was performed: *** p > 0.001; ** p < 0.01; * p < 0.05;. p < 0.1; n.s., not significant. For MS-data, ** FDR < 0.01. (C) WB images for pERK1 (pThr 203 /pTyr 205 ) or pERK2 (pThr 183 /pTyr 185 ) and total ERK1 and ERK2. Analyses were performed using three independent biological replicates of Bic-treated, TTX-treated, and untreated (control) neurons for 5 min (left) or 24 h (right). (D) Bar graphs show quantification of pERK1 and pERK2 detected by WB in comparison to the phosphopeptides quantified via

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Starvation induces CAMK2A-MLKL association in a protein complex. (A and B) Cell lysates from N2a cells untreated or treated with ECS for 1 h were subject to immunoprecipitation (IP) by IgG, or anti-CAMK2A (A) or anti-MLKL (B) antibody, followed by western blotting for MLKL and CAMK2A. (M) and (rb) denote the antibody host species as mouse and rabbit, respectively. The reciprocal co-immunoprecipitation showed increased association between CAMK2 and MLKL following starvation. (C) As in ( A ) but the cells were treated with KN93 (10 uM) as indicated. The blot by the mouse anti-CAMK2A antibody (CAMK2A[m]) shows the presence of antibody heavy chain in IP products from both IgG(m) and CAMK2A(m). However, the rabbit anti-CAMK2A antibody (CAMK2A[rb]) only revealed bands in this area for IP products from CAMK2A(m), but not that from IgG(m), indicating no cross reactivity of the secondary antibody. (D) In vitro kinase assay showing MLKL phosphorylation by recombinant CAMK2A. HA-MLKL purified by IP from transfected N2a cells was incubated with CALM, CAMK2A and ATP as indicated in a kinase buffer. The samples were subject to western blotting by the p-MLKL antibody. HA and ACTB were also measured from the cell lysates.