Cmpk1 Antibody

What is CMPK1 and why is it important in research?

CMPK1 (Cytidine Monophosphate (UMP-CMP) Kinase 1, Cytosolic) is an enzyme that catalyzes the phosphorylation of pyrimidine nucleoside monophosphates, particularly UMP and CMP, at the expense of ATP. It plays a crucial role in the formation of UDP, CDP, and dCDP, which are required for cellular nucleic acid synthesis . The enzyme belongs to the adenylate kinase family and has emerged as an important research target due to its role in nucleotide metabolism and its reported significance as a prognostic marker for several cancers including non-small cell lung cancer, pancreatic cancer, and breast cancer .

The protein exists in multiple isoforms with molecular weights of approximately 22-26 kDa, with the longest deduced protein containing 228 amino acids . Understanding CMPK1 function provides insights into fundamental cellular processes and potential disease mechanisms, making CMPK1 antibodies valuable tools for basic and translational research.

What are the different types of CMPK1 antibodies available for research applications?

CMPK1 antibodies are available in several formats to accommodate different experimental needs:

The choice between these antibody types depends on the specific research question, with polyclonal antibodies offering broader epitope recognition and monoclonal antibodies providing higher specificity. Monoclonal antibodies like clone 2D11G7 have been validated extensively for applications such as Western blotting with dilutions ranging from 1:5000-1:50000 .

How do I determine the appropriate molecular weight for CMPK1 detection?

When detecting CMPK1 using antibody-based methods, researchers should be aware of the multiple isoforms that can be observed:

• The full-length protein contains 228 amino acids with a calculated molecular weight of 26 kDa

• Translation likely begins at the second initiation methionine, resulting in a shorter form of 196 amino acids that represents the endogenously translated protein

• CMPK1 has been observed in 3 isoforms with molecular masses of 22, 16, and 26 kDa

When performing Western blot analysis, expect to observe bands primarily in the 22-26 kDa range, as documented in validated antibody applications . If you observe unexpected band sizes, consider using positive controls such as HeLa, HEK-293, or Jurkat cell lysates, which have been confirmed to express detectable levels of CMPK1 .

What are the recommended protocols for CMPK1 antibody applications in different techniques?

Different experimental techniques require specific optimization approaches for CMPK1 detection:

Western Blotting (WB):

• Sample preparation: NETN lysis buffer is recommended for effective protein extraction

• Antibody dilutions: For monoclonal antibodies, 1:5000-1:50000; for polyclonal antibodies, typically 0.1 μg/mL

• Positive controls: HeLa, HEK-293T, and Jurkat cell lysates (typically load 50 μg of total protein)

Immunofluorescence (IF/ICC):

• Fixation: 4% paraformaldehyde at room temperature for 15 minutes

• Antibody dilution: 1:200-1:800 for unconjugated antibodies; 1:50-1:500 for fluorescent-conjugated antibodies

• Counterstain: Hoechst 33342 for nuclear visualization

ELISA:

• Follow standard indirect ELISA protocols with antibody dilutions determined through titration experiments

• Validate specificity using recombinant CMPK1 protein (particularly the 37-196AA region)

Regardless of application, it is recommended to titrate the antibody concentration in each testing system to obtain optimal results and validate using appropriate positive controls .

How should I select between monoclonal and polyclonal CMPK1 antibodies for my research?

The selection between monoclonal and polyclonal CMPK1 antibodies depends on your specific experimental objectives:

For detecting CMPK1 in human samples, both types have been validated successfully. Consider monoclonal antibodies like clone 2D11G7 for applications requiring high reproducibility , while polyclonal antibodies may be advantageous for initially confirming expression in new experimental systems or when working with challenging samples .

What controls should I include when using CMPK1 antibodies?

Proper controls are essential for validating CMPK1 antibody results:

Positive Controls:

• Cell lines: HeLa, HEK-293, Jurkat cells have confirmed CMPK1 expression

• Tissue samples: Mouse brain has been validated as a positive control for CMPK1 expression

Negative Controls:

• Omission of primary antibody to assess background from secondary antibody

• Isotype control (matching IgG) to evaluate non-specific binding

• If available, CMPK1 knockout or knockdown samples provide definitive negative controls

Specificity Controls:

• Pre-absorption with immunogen peptide (particularly the recombinant fragments used as immunogens, such as aa 37-196)

• Comparison of staining patterns across multiple antibodies targeting different CMPK1 epitopes

Including these controls in your experimental design will strengthen the validity of your findings and help troubleshoot any unexpected results.

How can I distinguish between CMPK1 isoforms in my experiments?

Distinguishing between CMPK1 isoforms requires careful experimental design:

• Select appropriate antibodies: Choose antibodies with known epitope specificity. For example, antibodies targeting the N-terminal region (aa 1-37) would detect the full-length 26 kDa isoform but not the 22 kDa isoform that begins at the second methionine .

• Use high-resolution gel systems: Employ gradient gels (10-15%) with extended run times to achieve better separation of the 22, 16, and 26 kDa isoforms.

• Perform 2D-gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate isoforms that may have similar molecular weights but different post-translational modifications.

• Consider mass spectrometry validation: For definitive isoform identification, extract bands of interest and perform mass spectrometry to identify specific peptide sequences unique to each isoform.

• Implement isoform-specific RT-PCR: Design primers targeting unique regions of each isoform's mRNA to assess transcriptional expression patterns before protein analysis.

The scientific literature indicates that the shorter 22 kDa form (196 amino acids) is the predominant endogenously translated protein rather than the full-length 228 amino acid (26 kDa) protein , which is important to consider when interpreting experimental results.

What is the relationship between CMPK1 expression and cancer, and how can antibodies help study this correlation?

CMPK1 has been identified as a prognostic marker for several cancers, including non-small cell lung cancer, pancreatic cancer, and breast cancer . CMPK1 antibodies provide valuable tools for investigating these correlations through:

• Tissue microarray analysis: Using IHC with CMPK1 antibodies to assess expression patterns across tumor samples and correlate with clinical outcomes.

• Cancer cell line characterization: Western blotting and ICC/IF to compare CMPK1 expression levels across different cancer cell lines (such as HCT 116 colorectal carcinoma ).

• Subcellular localization studies: IF/ICC to determine whether CMPK1 localization changes during cancer progression, which might provide insights into altered protein function.

• Functional studies: Combining CMPK1 antibodies with other research tools (such as siRNA knockdown) to investigate the mechanistic role of CMPK1 in cancer development.

When designing such studies, researchers should consider using multiple detection methods and antibodies targeting different CMPK1 epitopes to strengthen the validity of their findings and account for potential isoform-specific effects.

How do CMPK1 antibodies perform in detecting cross-species reactivity?

CMPK1 is highly conserved across species, allowing many antibodies to detect the protein in multiple organisms:

When working with non-human samples, consider:

• Examining sequence homology in the region of the immunogen used to generate the antibody

• Starting with higher antibody concentrations for initial testing

• Validating with appropriate positive controls from the species of interest

• Using polyclonal antibodies for initial cross-species detection, as they may recognize multiple epitopes

For example, the polyclonal antibody PA5-28268 has predicted reactivity with mouse (99%), rat (98%), Xenopus laevis (85%), chicken (91%), rhesus monkey (100%), and bovine (98%) , making it potentially useful for comparative studies across species.

How should I troubleshoot weak or absent CMPK1 signal in Western blot experiments?

When encountering weak or absent CMPK1 signals in Western blotting, consider these methodological solutions:

• Sample preparation issues:

  • Ensure complete protein extraction using recommended buffers like NETN

  • Avoid repeated freeze-thaw cycles of protein samples

  • Include protease inhibitors in lysis buffers

• Protein degradation concerns:

  • Check protein integrity by staining the membrane with Ponceau S

  • Verify appropriate sample handling and storage (-80°C for long-term)

  • Use freshly prepared samples when possible

• Technical optimization:

  • Increase antibody concentration (start with 1:1000 dilution for initial troubleshooting)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different blocking agents (5% non-fat milk vs. 3-5% BSA)

  • Optimize transfer conditions for proteins in the 22-26 kDa range

• Antibody-specific solutions:

  • If using a monoclonal antibody with weak signal, try a polyclonal alternative

  • Confirm antibody storage conditions (avoid repeated freeze-thaw cycles)

  • Verify epitope accessibility; some epitopes may be masked by sample preparation methods

• Expression verification:

  • Confirm CMPK1 mRNA expression in your samples via RT-PCR

  • Include positive control lysates (HeLa, HEK-293, or Jurkat cells)

These methodological approaches should help resolve common technical issues with CMPK1 detection.

What are the best practices for quantitative analysis of CMPK1 expression?

For accurate quantitative analysis of CMPK1 expression, researchers should follow these methodological best practices:

• Western blot quantification:

  • Use appropriate loading controls (β-actin, GAPDH, or α-tubulin)

  • Ensure signal is within the linear range of detection

  • Analyze multiple biological replicates (minimum n=3)

  • Use validated software (ImageJ, Image Studio, etc.) for densitometry

  • Apply statistical analysis to determine significance

• Immunofluorescence quantification:

  • Standardize image acquisition parameters across all samples

  • Collect multiple fields (>5) per condition

  • Use nuclear counterstains for cell number normalization

  • Apply automated analysis with consistent thresholding

  • Consider single-cell analysis for populations with heterogeneous expression

• ELISA-based quantification:

  • Generate standard curves using recombinant CMPK1 protein

  • Include technical replicates (minimum duplicates)

  • Ensure sample concentrations fall within the linear range of the standard curve

• Cross-validation:

  • Validate findings using at least two different antibodies

  • Confirm protein expression changes with mRNA analysis

  • Consider functional assays to correlate expression with activity

These approaches will enhance the reproducibility and reliability of quantitative CMPK1 expression analyses across different experimental systems.

How do I interpret contradictory results from different CMPK1 antibodies?

When different CMPK1 antibodies yield contradictory results, follow this methodological framework for interpretation:

• Evaluate antibody characteristics:

  • Compare immunogen regions (epitopes) targeted by each antibody

  • Assess whether antibodies recognize different isoforms

  • Check validation data from manufacturers for expected patterns

• Consider technical variations:

  • Different antibody classes (monoclonal vs. polyclonal) may yield different results

  • Various applications (WB vs. IF/ICC) may have different sensitivities

  • Sample preparation methods can affect epitope accessibility

• Cross-validation approaches:

  • Perform knockdown/knockout experiments to confirm specificity

  • Use peptide competition assays with the immunizing peptide

  • Compare with published literature on expected CMPK1 expression patterns

  • Verify with orthogonal techniques (e.g., mass spectrometry)

• Isoform-specific considerations:

  • CMPK1 exists in multiple isoforms (22, 16, and 26 kDa)

  • Antibodies targeting different regions may detect specific isoforms

  • The shorter 196 amino acid form is the predominant endogenously translated protein

• Application-specific interpretation:

  • For Western blotting: Focus on antibodies with clear bands at expected molecular weights

  • For ICC/IF: Evaluate subcellular localization consistency with known CMPK1 biology

  • For IHC: Compare with established expression patterns in relevant tissues

By systematically analyzing these factors, researchers can reconcile contradictory results and determine the most reliable data for their experimental system.

What are emerging research areas involving CMPK1 antibodies?

CMPK1 antibodies are increasingly being utilized in several emerging research areas:

• Cancer biomarker development:

  • Evaluating CMPK1 as a prognostic marker in additional cancer types beyond the established associations with non-small cell lung cancer, pancreatic cancer, and breast cancer

  • Investigating correlations between CMPK1 expression patterns and treatment response

• Drug development applications:

  • Studying CMPK1's role in activating nucleoside analog drugs

  • Evaluating potential therapeutic targeting of CMPK1 in cancer therapy

  • Monitoring CMPK1 expression changes in response to treatment

• Systems biology approaches:

  • Integration of CMPK1 expression data with other omic datasets

  • Network analysis of CMPK1's role in pyrimidine metabolism pathways

  • Correlation of CMPK1 activity with cellular metabolic states

• Development of advanced detection methods:

  • Multiplexed immunoassays including CMPK1 with other biomarkers

  • Single-cell analysis of CMPK1 expression heterogeneity

  • In vivo imaging applications with conjugated CMPK1 antibodies

These emerging areas highlight the continuing importance of high-quality CMPK1 antibodies for advancing our understanding of nucleotide metabolism in health and disease.

What methodological advances are improving CMPK1 antibody applications?

Recent methodological advances are enhancing the utility of CMPK1 antibodies in research:

• Conjugated antibody technologies:

  • Directly conjugated fluorescent CMPK1 antibodies (like CoraLite® Plus 488) enable direct visualization without secondary antibodies

  • Multiplexed detection systems allow simultaneous visualization of CMPK1 with other proteins

• Improved validation standards:

  • Application-specific validation using knockout/knockdown systems

  • Cross-platform verification (e.g., correlating results from Western blot, IHC, and MS)

  • Standardized reporting of validation data by antibody manufacturers

• Advanced imaging applications:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging techniques for dynamic CMPK1 studies

  • Automated high-content analysis for screening applications

• Proximity-based assays:

  • Proximity ligation assays (PLA) to study CMPK1 protein-protein interactions

  • FRET-based approaches to monitor CMPK1 activity in living cells

• Computational prediction and analysis:

  • Improved epitope prediction algorithms for antibody design

  • Machine learning approaches for analysis of CMPK1 expression patterns in complex datasets