PRKCA Antibody
Description
Overview of PRKCA and Its Antibodies
PRKCA encodes protein kinase C alpha (PKCα), a member of the conventional PKC family activated by calcium and diacylglycerol. It regulates signal transduction pathways implicated in cancer, neurological disorders, and cardiovascular diseases . PRKCA antibodies are immunoglobulin-based reagents designed to bind specifically to PRKCA for applications such as:
Western Blotting
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Boster Bio’s PA1065 detects PRKCA at ~77 kDa in human U87 and HEL cell lysates, rat/mouse brain lysates .
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Cell Signaling Tech’s #4782 identifies endogenous PRKCA at 42 kDa in human, mouse, and rat samples .
Immunohistochemistry
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PA1065 demonstrates strong staining in human mammary cancer tissue sections, validated with DAB chromogen and peroxidase-based detection .
Functional Insights
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PRKCA antibodies have revealed the protein’s role in stabilizing VEGFA mRNA (promoting angiogenesis) and regulating platelet aggregation via ITGA2B-ITGB3 integrin activation .
Disease Relevance
PRKCA dysregulation is linked to:
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Cancer: Overexpression in breast, lung, and hematologic malignancies .
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Neurological Disorders: Altered signaling in Alzheimer’s and Parkinson’s diseases .
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Male Infertility: Associated with globozoospermia due to defective spermatogenesis .
Validation and Quality Control
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Specificity: Boster Bio’s PA1065 uses a synthetic peptide immunogen unique to human PRKCA (one amino acid difference from rodent sequences) .
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Cross-Reactivity: No cross-reactivity with other proteins reported for PA1065 .
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Storage: Lyophilized antibodies are stable at -20°C for 1 year; reconstituted variants last 1 month at 4°C .
Limitations and Considerations
Product Specs
Target Background
PRKCA's influence on cell proliferation is complex, demonstrating both positive and negative regulatory functions during the cell cycle. It can promote cell growth by phosphorylating and activating RAF1, leading to the activation of the MAPK/ERK signaling cascade. Additionally, PRKCA upregulates CDKN1A, facilitating the formation of active cyclin-dependent kinase (CDK) complexes in glioma cells. Notably, in intestinal cells stimulated by the phorbol ester PMA, PRKCA can trigger cell cycle arrest, a process associated with the accumulation of the hyper-phosphorylated growth-suppressive form of RB1 and the induction of CDK inhibitors CDKN1A and CDKN1B.
PRKCA exhibits anti-apoptotic functions in various cell types. In glioma cells, it protects against apoptosis by suppressing the p53/TP53-mediated activation of IGFBP3. In leukemia cells, it mediates anti-apoptotic action through the phosphorylation of BCL2.
During macrophage differentiation induced by macrophage colony-stimulating factor (CSF1), PRKCA translocates to the nucleus and is linked to macrophage development. In response to wounding, PRKCA relocates from focal contacts to lamellipodia and participates in the modulation of desmosomal adhesion. Its role in cell motility is demonstrated by its phosphorylation of CSPG4, which induces association with extensive lamellipodia at the cell periphery and polarization of the cell, leading to increased motility. In chemokine-induced CD4(+) T cell migration, PRKCA phosphorylates CDC42-guanine exchange factor DOCK8, causing its dissociation from LRCH1 and the activation of GTPase CDC42.
PRKCA is highly expressed in many cancer cells where it can act as a tumor promoter, implicated in the malignant phenotypes of various tumors such as gliomas and breast cancers. It negatively regulates myocardial contractility while positively regulating angiogenesis, platelet aggregation, and thrombus formation in arteries. PRKCA mediates the hypertrophic growth of neonatal cardiomyocytes, partly through a MAPK1/3 (ERK1/2)-dependent signaling pathway. Upon PMA treatment, it is essential for inducing cardiomyocyte hypertrophy, leading to heart failure and death by increasing protein synthesis, protein-DNA ratio, and cell surface area. PRKCA regulates cardiomyocyte function by phosphorylating cardiac troponin T (TNNT2/CTNT), which significantly reduces actomyosin ATPase activity, myofilament calcium sensitivity, and myocardial contractility.
In angiogenesis, PRKCA is crucial for full endothelial cell migration, adhesion to vitronectin (VTN), and vascular endothelial growth factor A (VEGFA)-dependent regulation of kinase activation and vascular tube formation. It is involved in stabilizing VEGFA mRNA at the post-transcriptional level and mediates VEGFA-induced cell proliferation.
Regarding calcium-induced platelet aggregation, PRKCA mediates signals from the CD36/GP4 receptor for granule release and activates the integrin heterodimer ITGA2B-ITGB3 through the RAP1GAP pathway for adhesion. In response to lipopolysaccharides (LPS), PRKCA may regulate selective LPS-induced macrophage functions involved in host defense and inflammation. However, in certain inflammatory responses, it may negatively regulate NF-kappa-B-induced genes through IL1A-dependent induction of NF-kappa-B inhibitor alpha (NFKBIA/IKBA). Upon stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), PRKCA phosphorylates EIF4G1, which modulates EIF4G1 binding to MKNK1 and may be involved in the regulation of EIF4E phosphorylation. It also phosphorylates KIT, leading to inhibition of KIT activity, and ATF2, promoting cooperation between ATF2 and JUN, activating transcription. Furthermore, PRKCA phosphorylates SOCS2 at 'Ser-52', facilitating its ubiquitination and proteosomal degradation.
- The D463H mutation in PRKCA, highly specific to chordoid glioma, enhances proliferation of astrocytes and tanycytes. PMID: 29915258
- Modeling of the different conformations of PRKACA-DNAJB1 Chimeric Kinase revealed no obvious steric interactions of the J-domain with the rest of the RIIbeta holoenzyme. PMID: 29335433
- PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. PMID: 28186199
- Protein kinase C acts as a tumor suppressor. Cancer-associated mutations in protein kinase C are generally loss-of-function mutations.[review] PMID: 28476658
- These results could not only better explain the role of PI-PLCbeta1/PKC-alpha signaling in erythropoiesis but also lead to a better comprehension of the lenalidomide effect on del(5q) MDS and pave the way to innovative, targeted therapies. PMID: 28970249
- A characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKCalpha activation. Moreover, endocytosis of ATP11C is induced by Ca(2+)-signaling via Gq-coupled receptors. PMID: 29123098
- The haplotype carrying rs9909004 influences PRKCA expression in the heart and is associated with traits linked to heart failure, potentially affecting therapy of heart failure. PMID: 28120175
- Our results demonstrate that Pc-induced expression of HO-1 is mediated by the PKCA-Nrf-2/HO-1 pathway, and inhibits UVB-induced apoptotic cell death in primary skin cells. PMID: 29470442
- Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCalpha signalling and cross-talk with TGF-beta1. PMID: 29016732
- This study reveals a protective role for miR-706 by blocking the oxidative stress-induced activation of PKCalpha/TAOK1. Our results further identify a major implication for miR-706 in preventing hepatic fibrogenesis and suggest that miR-706 may be a suitable molecular target for anti-fibrosis therapy. PMID: 27876854
- We also discuss the contribution of PKC enzymes to pancreatic diseases, including insulin resistance and diabetes mellitus, as well as pancreatitis and the development and progression of pancreatic cancer. PMID: 28826907
- Data provided evidence that increased Rack1-mediated upregulation of PKC kinase activity may be responsible for the development of chemoresistance in T-ALL-derived cell line potentially by reducing FEM1b and Apaf-1 level. PMID: 27644318
- Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells has been summarized. (Review) PMID: 29029784
- These data propose a mechanism where CD82 membrane organization regulates sustained PKCalpha signaling that results in an aggressive leukemia phenotype. These observations suggest that the CD82 scaffold may be a potential therapeutic target for attenuating aberrant signal transduction in acute myeloid leukemia (AML). PMID: 27417454
- MiR-3148 may play an important role in the development of CTEPH. The key mechanisms for this miRNA may be hsa-miR-3148-AR-pathways in cancer or hsa-miR-3148-PRKCA-pathways in cancer/glioma/ErbB signaling pathway. PMID: 28904974
- The spatial organization of cPKCs bound to the plasma membrane is reported. PMID: 27808106
- PRKCA is a recurrently mutated oncogene in human chordoid glioma. PMID: 29476136
- Our study showed that PKCalpha modulated cell resistance to apoptosis by stimulating NF-kappaB activation and thus promoted the tumorigenesis of bladder cancer. PMID: 28629334
- PKCalpha translocation may occur as an early event in radiation-induced bystander responses. PMID: 27165942
- Our study indicated that PKC alpha and beta appeared coping with oncogenic Ras or mutated Akt to maintain the balance of the homeostasis in cancer cells. Once these PKC isoforms were suppressed, the redox state in the cancer cells was disrupted, which elicited persistent oncogenic stress and subsequent apoptotic crisis. PMID: 28415683
- High expression of both PLCE1 and PRKCA is significantly associated with poor outcomes of the patients with esophageal cancers. PMID: 28402280
- In nasopharyngeal carcinoma, PKCalpha is linked to the invasion of adjacent tissues, especially in the skull base. Down-regulation of PKCalpha is a risk factor for regional lymph node metastasis. PMID: 28084179
- LAV-BPIFB4 isoform modulates eNOS signalling through Ca2+/PKC-alpha-dependent mechanism. PMID: 28419216
- Studied interactions between protein kinase C alpha (PKCalpha), FOXC2, and p120-catenin (CTNND1) in breast cancer, cell migration/ invasion; found PKCalpha acts as an upstream regulator of FOXC2, which in turn represses the expression of p120-catenin, in both in endocrine resistant ER+breast cancer and basal A triple negative breast cancer. PMID: 29216867
- Phosphorylated PKCalpha is elevated in epidermis genetically deleted of DLX3 and the hyperproliferative response to TPA is increased, suggesting that the homeobox protein indirectly regulates the activity in the pathway, possibly through an effect on reduced phosphatase expression. PMID: 28186503
- Results show that PKCalpha expression is under the regulation of miR-142-3p contributing to reduced osteoclasts survival. PMID: 27113904
- Molecular Determinants for the Binding Mode of Alkylphosphocholines in the C2 Domain of PKCalpha. PMID: 27490031
- Studies suggest that rare deleterious variants of PARD3 in the aPKC-binding region contribute to human cranial neural tube defect (NTD). PMID: 27925688
- This study identified PKCalpha as hepatitis E virus HEV in host defense. PMID: 28077314
- ADP inhibits mesothelioma cell proliferation via PKC-alpha/ERK/p53 signaling. PMID: 28777435
- This study provides evidences of a new PKCalpha/GAP-43 nuclear signalling pathway that controls neuronal differentiation in Human Periodontal Ligament Stem Cells. PMID: 27478064
- Protein kinase Calpha (PKCalpha) gain of function mutations may promote synaptic defects in Alzheimer's disease. PMID: 27165780
- Some polyphenols exert their antioxidant properties by regulating the transcription of the antioxidant enzyme genes through PKC signaling. Regulation of PKC by polyphenols is isoform dependent. PMID: 27369735
- Data suggest that phosphorylation activity of PRKCA stems from conformational flexibility in region C-terminal to phosphorylated Ser/Thr residues; flexibility of substrate-kinase interaction enables an Arg/Lys two to three amino acids C-terminal to phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to substrate. PMID: 28821615
- These results provide evidence for inherent deficits in the cystic fibrosis macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia. PMID: 28093527
- The interplay between intracellular progesterone receptor and PRKCA-PRKCD plays a key role in migration and invasion of human glioblastoma cells. PMID: 27717886
- PRKCA SNPs are associated with neuropathic pain post total joint replacement. PMID: 28051079
- These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca(2+) influx via L-type voltage-sensitive Ca(2+) channels as a mechanism for histamine-induced bronchoconstriction. PMID: 28283479
- Pseudosubstrate and C1a domains, however, are minimally essential for maintaining the inactivated state. Furthermore, disrupting known interactions between the C1a and other regulatory domains releases the autoinhibited interaction and increases basal activity. PMID: 28049730
- In polymorphism PRKCA rs9892651, HDL-C levels were lower in carriers of CC and TC genotypes that were more frequent in current-wheezers Vs TT genotype (52.2 and 52.7 Vs 55.2 mg/dl, p-value = 0.042 and p-value for trend = 0.02). PMID: 27411394
- Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro. PMID: 27119641
- Phosphorylation of TIMAP on Ser331 by PKC represents a new mechanism of endothelial barrier regulation, through the inhibition of phospho-ERM dephosphorylation. PMID: 27939168
- PKCalpha-GSK3beta-NF-kappaB signaling pathway involvement in TRAIL-induced apoptosis. PMID: 27219672
- Curcumin inhibited phorbol ester-induced membrane translocation of protein kinase C-epsilon (PKCepsilon) mutants, in which the epsilonC1 domain was replaced with alphaC1, but not the protein kinase C-alpha (PKCalpha) mutant in which alphaC1 was replaced with the epsilonC1 domain, suggesting that alphaC1 is a determinant for curcumin's inhibitory effect. PMID: 27776404
- A library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Calpha and 14 substrate peptides. PMID: 27555323
- Calpain and protein kinase Calpha abnormal release promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients. PMID: 27349634
- Protein kinase C modulates alpha1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off. PMID: 28082304
- Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes. PMID: 27376808
- These results confirm the correlation between AXL and PKCalpha, and suggest PKCalpha-AXL signaling may be a treatment target, particularly in malignant cancer cells. PMID: 27357025
- After inhibition of the PKC/ERK signalling pathway, the effects of DOR on breast cancer were significantly attenuated in vivo and in vitro. In summary, DOR is highly expressed in breast cancer and is closely related to its progression. These results suggest that DOR may serve as a potential biomarker for the early diagnosis of breast cancer and may be a viable molecular target for therapeutic intervention. PMID: 27665747
Q&A
What is PRKCA and what cellular functions does it regulate?
PRKCA is a calcium-independent, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase belonging to the PKC family. This kinase plays critical roles in multiple cellular processes including cell adhesion, cell transformation, cell cycle checkpoint regulation, and cell volume control. PRKCA phosphorylates various protein targets and participates in diverse cellular signaling pathways . Knockout studies in mice suggest that PRKCA functions as a fundamental regulator of cardiac contractility and calcium handling in myocytes .
PKC family members serve as major receptors for phorbol esters (a class of tumor promoters) and mediate important functions in cell signaling cascades. Each member of the PKC family has a specific expression profile and is believed to play distinct roles in cellular processes .
How do I choose between polyclonal and monoclonal PRKCA antibodies?
The choice between polyclonal and monoclonal antibodies depends on your experimental requirements:
| Antibody Type | Advantages | Limitations | Ideal Applications |
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| Polyclonal | Recognizes multiple epitopes; Stronger signal; Better for detecting denatured proteins | Batch-to-batch variation; Potentially higher background | Western blotting; Immunoprecipitation |
| Monoclonal | Consistent specificity; Lower background; Better for distinguishing isoforms | May be sensitive to epitope modifications; Potentially weaker signal | Immunofluorescence; Flow cytometry; Detecting specific conformations |
For example, rabbit polyclonal antibodies against PRKCA are suitable for immunoprecipitation (IP) and Western blotting (WB) . When selecting an antibody, consider the nature of your sample (native vs. denatured protein) and your specific experimental requirements for specificity.
How should I validate the specificity of my PRKCA antibody?
Validation is critical for ensuring experimental reliability. Current best practices include:
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Genetic validation: Using CRISPR/Cas9-generated knockout cell lines provides the gold standard for antibody validation. A specific antibody should show signal in wild-type cells but not in knockout cells lacking the antigenic epitope .
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Pharmacological validation: Treat cells with known PKC activators such as phorbol 12-myristate 13-acetate (PMA) and inhibitors like Gö6983. Phospho-specific antibodies should show increased signal with activators and decreased signal with inhibitors .
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Phosphatase treatment: For phospho-specific antibodies or when phosphorylation may mask epitopes, in vitro phosphatase treatment can restore antibody recognition, confirming specificity .
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Multiple detection methods: Validate using different techniques (Western blot, immunofluorescence, flow cytometry) to ensure consistent detection across platforms.
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Cross-reactivity assessment: Test against related PKC isoforms to ensure isoform specificity.
What applications are PRKCA antibodies suitable for?
PRKCA antibodies can be used in various experimental applications:
Different antibodies may perform optimally in specific applications. For example, some antibodies specifically recognize the C2-domain of PKC and can detect active conformations in immunofluorescence applications .
What should I know about antibody epitope recognition and phosphorylation?
Phosphorylation can significantly affect antibody recognition of PRKCA:
How can I distinguish between different PKC isoforms using antibodies?
Distinguishing between closely related PKC isoforms requires careful antibody selection and validation:
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Isoform-specific epitopes: Select antibodies raised against unique regions that differ between PKC isoforms. The C-terminal region often contains isoform-specific sequences.
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Conformation-specific antibodies: Researchers have developed antibodies that specifically recognize the C2-domain of particular PKC isoforms (e.g., C2-Cat-PKCβ for PKCβ specifically, and C2-Cat-cPKC for all conventional PKCs) .
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Validation using knockout cells: To confirm specificity, test antibodies in cell lines with specific PKC isoforms knocked out. For example, C2-Cat-PKCβ antibody shows reduced fluorescence in PKCβ-KO cells but not in PKCα-KO cells .
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Pharmacological approach: Different PKC isoforms may respond differently to activators and inhibitors. For instance, conventional PKCs like PRKCA show increased detection with C2-Cat-cPKC antibody upon PMA treatment, and this signal is eliminated by PKC inhibitor Gö6983 .
What are conformation-specific antibodies for PKC and when should I use them?
Conformation-specific antibodies recognize particular structural configurations of proteins and are valuable tools for studying signaling dynamics:
How can genome editing techniques be used to validate PRKCA antibodies?
CRISPR/Cas9 genome editing provides a powerful approach for antibody validation:
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Knockout generation: Generate cell lines lacking PRKCA using CRISPR/Cas9 to create negative controls for antibody testing .
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Validation workflow:
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Combined analytical approaches: High-throughput microscopy (HTM) combined with genome editing provides quantitative assessment of antibody specificity. For example, researchers used HTM analysis to measure changes in fluorescence intensity in wild-type versus PKCα-KO or PKCβ-KO cells .
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Stimulus response in knockout models: Treating wild-type and knockout cells with PKC activators (PMA) further confirms antibody specificity. Phospho-specific antibodies should show increased signal in wild-type cells but not in knockout cells upon activation .
How do I optimize detection of active PRKCA in experimental systems?
Detecting active PRKCA requires specialized approaches targeting activation-dependent changes:
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Conformation-specific antibodies: Antibodies like C2-Cat-cPKC specifically recognize the active conformation of conventional PKCs including PRKCA. These show increased fluorescence upon PKC activation with PMA treatment .
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Experimental design considerations:
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Include both positive controls (PMA treatment, which activates PKC) and negative controls (PKC inhibitors like Gö6983)
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Monitor activation kinetics with time-course experiments
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Compare results across different cell types to account for context-dependent activation mechanisms
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Validation in multiple systems: Test activation detection in different cell types to confirm that the approach is not cell-type specific. For example, researchers demonstrated that C2-Cat-cPKC antibody detects PMA-induced PKC activation in both mouse Neuro2A and human SK-N-SH neuroblastoma cell lines .
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Inhibitor controls: Include specific PKC inhibitors to confirm that the detected signals are truly PKC-dependent. For example, the PMA-induced increase in C2-Cat-cPKC signal is completely eliminated by the PKC inhibitor Gö6983 .
What technical considerations affect interpretation of PRKCA antibody results?
Several technical factors can significantly impact PRKCA antibody results:
Why might my Western blot show unexpected bands with PRKCA antibodies?
Unexpected bands in Western blots can result from several factors:
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Protein processing: PRKCA can exist in multiple forms due to post-translational modifications. The predicted protein size for PRKCA ranges from 59-67kDa/74-83kDa depending on modifications .
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Cross-reactivity: Some antibodies may cross-react with other PKC isoforms due to sequence homology. Validation using knockout cell lines can help identify specific versus non-specific bands .
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Epitope masking: Phosphorylation can mask epitopes, resulting in reduced or absent bands despite protein presence. In vitro phosphatase treatment can restore detection if this is the case .
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Degradation products: Proteolytic degradation during sample preparation can generate fragments that are recognized by the antibody.
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Optimization strategies:
How can I accurately compare PRKCA levels between active and inactive states?
Comparing PRKCA levels between different activation states requires careful experimental design:
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Antibody selection: Use antibodies whose epitopes are not masked by phosphorylation events that occur during activation. Commercial antibodies may have uncharacterized recognition properties that change with protein phosphorylation .
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Multiple antibody approach: Use both phospho-specific antibodies and total protein antibodies whose epitopes are not affected by activation state.
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Control experiments:
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Complementary techniques: Combine immunoblotting with other techniques like mass spectrometry to comprehensively analyze protein levels and modifications.
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Careful interpretation: What appears as protein downregulation may actually be reduced antibody binding due to epitope masking by phosphorylation .
