PA5-17551

antibody from Invitrogen Antibodies

Targeting: PRKCA PKCA, PKCα

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Recommended by provider)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-17551

Provider

Invitrogen Antibodies

Product name

PKC alpha Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

Antibodies to this protein (and modification) were previously sold as part of a Thermo Scientific Cellomics High Content Screening Kit. This replacement antibody is now recommended for researchers who need an antibody for high content cell based assays. It has been thoroughly tested and validated for cellular immunofluorescence (IF) applications. Further optimization including the selection of the most appropriate fluorescent Dylight conjugated secondary antibody may have to be performed for your high content assay. It is not recommended to aliquot this antibody. This antibody is not cross-reactive with endogenous levels of other PKC isoforms.

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

11 µg/mL

Storage

-20°C

References

Submitted references

Cell signaling pathways in autosomal-dominant leukodystrophy (ADLD): the intriguing role of the astrocytes.

Ratti S, Rusciano I, Mongiorgi S, Owusu Obeng E, Cappellini A, Teti G, Falconi M, Talozzi L, Capellari S, Bartoletti-Stella A, Guaraldi P, Cortelli P, Suh PG, Cocco L, Manzoli L, Ramazzotti G
Cellular and molecular life sciences : CMLS 2021 Mar;78(6):2781-2795

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A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKCγ Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior.

Shimobayashi E, Kapfhammer JP
The Journal of neuroscience : the official journal of the Society for Neuroscience 2021 Mar 3;41(9):2053-2068

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PRKCA was performed with 10 µg of HeLa cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against PRKCA (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. PRKCA was detected at ~ 80 kDa using PRKCA Rabbit polyclonal antibody (Product # PA5-17551) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Rabbit (Product # 31461) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). PRKCA Antibody (Product # PA5-17551) confirms silencing of PRKCA expression.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PKC-alpha in extracts from Baculovirus expressed PKC isoforms using PKC-alpha polyclonal antibody (Product # PA5-17551) demonstrating the isoform-specificity of PKC-alpha antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of PKC alpha was achieved by transfecting NIH/3T3 cells with PKC alpha specific validated siRNAs (Silencer® select Product # s11094). Western blot analysis (Fig. a) was performed using whole cell extracts from the PKC alpha knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with PKC alpha Polyclonal Antibody (Product # PA5-17551, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PKC alpha.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of NIH/3T3 (Lane 1), Jurkat (Lane 2) and U-2 OS (Lane 3). The blot was probed with Anti-PKC alpha Polyclonal Antibody (Product # PA5-17551, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 80 kDa band corresponding to PKC alpha was observed across the cell lines tested.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PRKCA was performed with 10 µg of HeLa cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against PRKCA (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. PRKCA was detected at ~ 80 kDa using PRKCA Rabbit polyclonal antibody (Product # PA5-17551) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Rabbit (Product # 31461) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). PRKCA Antibody (Product # PA5-17551) confirms silencing of PRKCA expression.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PKC-alpha in extracts from C6 and NIH/3T3 cells using PKC-alpha polyclonal antibody (Product # PA5-17551).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of PKC alpha was performed using 70% confluent log phase NIH/3T3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with PKC alpha Polyclonal Antibody (Product # PA5-17551) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of PKC-alpha in C6 cells, TPA-treated, using a PKC-alpha polyclonal antibody (Product # PA5-17551) (green). Actin filaments are labeled with a fluorescent red phalloidin. DNA is labeled using a fluorescent blue dye.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of PKC-alpha in C6 cells, serum-starved, using a PKC-alpha polyclonal antibody (Product # PA5-17551) (green). Actin filaments are labeled with a fluorescent red phalloidin. DNA is labeled using a fluorescent blue dye.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometric analysis of PKC-alpha in untreated 293 cells using a PKC-alpha polyclonal antibody (Product # PA5-17551) (blue) compared to a nonspecific negative control antibody (red).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Antibody specificity is demonstrated by a recombinant protein array. Western blotting of recombinant human PKC alpha and Beta proteins using PKC alpha Antibody (Product # PA5-17551) demonstrates isoform-specificity of the PKC-alpha antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3. Generation and characterization of the PKC gamma -A24E mouse. A , Target sequence in the PRKCG gene (chromosome 7, 1.93 cM) for producing point mutated (c. 71C > A; p. Ala 24 Glu) PRKCG with Cas9/CRISPR engineering system. To prevent donor DNA cleavage, the PAM sequence mutation was also introduced (78G > A), which does not give a change in amino acids. B , Mutations were validated with sequencing. The figure shows Het PKC gamma -A24E sequence. C , To identify the genotypes, PCR was performed followed by Alu1 (BioLabs, R0137S) digestion for 1 h, which digests only Ala 24 Glu mutated DNA. Wt shows a single band (250 bp), Het PKC gamma -A24E shows a Wt band and two digested bands (250, 150, and 100 bp), and Homo PKC gamma -A24E shows only two digested bands (150 and 100 bp). D , qPCR using 5-week-old cerebellar samples from each genotype. Data show that all three genotypes express PKCgamma mRNA. E , Western blot analysis of total PKCgamma protein in the cerebellum from the three genotypes ( n = 4). PKCgamma protein degradation in PKC gamma -A24E mice was observed (4-week-old: Wt = 100.0 +- 8.34%, n = 4; Het = 49.12 +- 6.80%, n = 4; Homo = 7.519 +- 2.48%, p = 0.0286, n = 4; 6-week-old: Wt = 100.0 +- 14.56%, n = 4; Het = 63.09 +- 6.39%, n = 4; Homo = 4.674 +- 0.40%, p = 0.0286, n = 4), whereas PKCalpha protein expression did not change in all genotypes (4-week-old: Wt = 100.0 +- 9.30%, n = 4; Het = 83.13 +- 2.09%, n = 4; Homo = 87.67 +- 14.1%, n = 4; 6-week-old: Wt = 10

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 3 Lamin B1 build up affects the expression and phosphorylation of signaling molecules downstream LIF-R. The expression and the phosphorylation of molecules downstream LIF- signaling pathway was evaluated in U87-MG ( a ) and MO3.13 ( b ) cells 96 h after transduction. Lamin B1 overexpressing cells ( ovLMNB1 ) were compared to wild type ( WT ) and mock-transduced ( GFP ) samples. The expression or phosphorylation of the denoted proteins was assayed. beta-tubulin was used as loading control. Western blot results are representative of three independent experiments