CCNL2 Antibody

What is CCNL2 and what cellular functions does it perform?

CCNL2 (Cyclin L2) belongs to the cyclin family, specifically the Cyclin L subfamily. It functions primarily as a transcriptional regulator that participates in regulating pre-mRNA splicing processes. Additionally, CCNL2 modulates the expression of critical apoptotic factors, potentially influencing cell death pathways .

The protein co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and associates with hyperphosphorylated (but not hypophosphorylated) RNA polymerase II and CDK p110 PITSLRE kinase via its N-terminal cyclin domains . This interaction pattern suggests a role in coupling transcription with RNA processing.

CCNL2 is expressed ubiquitously in normal human tissues and tumor cells, indicating its fundamental role in cellular processes . The protein is also known by several alternative names, including SB138, PCEE (Paneth cell-enhanced expression protein), CCNM, and others .

What is the molecular structure and characteristics of CCNL2 protein?

CCNL2 has a calculated molecular weight of approximately 58 kDa, though interestingly, the observed molecular weight in some Western blot applications is around 28 kDa . This discrepancy may result from:

• Post-translational modifications

• Alternative splicing events generating different isoforms

• Proteolytic processing in vivo or during sample preparation

• Anomalous migration on SDS-PAGE due to protein structure

The human CCNL2 protein is encoded by the gene located at GenBank accession number BC016333 (NCBI Gene ID: 81669) . The UniProt ID for human CCNL2 is Q96S94 .

Multiple isoforms of CCNL2 exist, with at least two documented variants: CCNL2a and CCNL2b. Some antibodies are specifically designed to detect only certain isoforms, such as those that target CCNL2a but show negligible reactivity to CCNL2b or related Cyclin L1 isoforms .

How do I select the appropriate CCNL2 antibody for my research application?

When selecting a CCNL2 antibody, consider these critical factors:

Target region specificity:

• N-terminal antibodies: Often useful for detecting full-length protein

• C-terminal antibodies: May help identify specific isoforms or processed forms

• Internal domain antibodies: Can provide information about protein folding or domain exposure

Application compatibility:
Different antibodies show varying performance across applications:

Species reactivity:
Most CCNL2 antibodies react with human CCNL2, with many also cross-reacting with mouse and rat orthologs . Verify species compatibility before use in your experimental system.

Validation documentation:
Look for antibodies with published validation data, including positive Western blot results in relevant cell lines (e.g., A375, HeLa, U-251 cells, human placenta tissue) .

What validation approaches should I use to confirm CCNL2 antibody specificity?

A comprehensive validation strategy should include:

• Knockdown/knockout controls:

  • siRNA/shRNA knockdown of CCNL2

  • CRISPR-Cas9 knockout cells (if available)

  • Compare results between treated and untreated samples

• Multiple antibody approach:

  • Use at least two different antibodies targeting distinct epitopes

  • Compare recognition patterns to confirm target identity

• Western blot analysis:

  • Verify expected molecular weight (accounting for the 58 kDa calculated vs. 28 kDa observed discrepancy)

  • Check for specificity across multiple cell lines with known CCNL2 expression levels

  • Use positive controls such as A375, HeLa, or U-251 cells

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with the CCNL2 antibody

  • Confirm protein identity using mass spectrometry

  • Analyze co-precipitating partners for expected interactions (e.g., splicing factors)

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Confirm signal reduction/elimination in appropriate assays

What are the key considerations for immunoprecipitation experiments with CCNL2 antibodies?

Protocol optimization:

• Use 1.0 mg of whole cell lysate per IP reaction

• For immunoblotting the immunoprecipitated CCNL2, a concentration of 0.04 μg/ml has been effective

• Load approximately 20% of the IP sample for subsequent analysis

Buffer systems:

• NETN lysis buffer has been demonstrated effective for CCNL2 immunoprecipitation from HeLa cells

Antibody amounts:

• 6 μg of antibody per reaction has successfully immunoprecipitated CCNL2 from cell lysates

• Affinity-purified antibodies generally show better performance than crude sera

Controls to include:

• IgG control (same species as the CCNL2 antibody)

• Input sample (typically 5% of starting material)

• Known CCNL2-interacting proteins as positive controls (e.g., splicing factors SC-35 and 9G8, CDK p110 PITSLRE)

Downstream applications:

• For co-immunoprecipitation studies, look for interactions with RNA polymerase II and splicing machinery components

• Consider mass spectrometry analysis to identify novel interaction partners

How can CCNL2 antibodies be used to investigate pre-mRNA splicing mechanisms?

CCNL2's role in pre-mRNA splicing can be studied through several methodological approaches:

Co-localization studies:

• Use fluorescently labeled CCNL2 antibodies alongside markers for nuclear speckles

• Track co-localization with established splicing factors such as SC-35 and 9G8

• Quantify spatial relationships during different stages of the cell cycle or after treatment with splicing modulators

Chromatin immunoprecipitation (ChIP):

• Use CCNL2 antibodies to precipitate chromatin-associated complexes

• Analyze co-precipitating DNA to identify genomic regions where CCNL2 is involved in transcription/splicing

• Compare with RNA Pol II ChIP data to understand temporal coordination

RNA immunoprecipitation (RIP):

• Immunoprecipitate CCNL2-RNA complexes

• Sequence associated RNAs to identify target transcripts

• Analyze for common sequence motifs or structural features

Splicing assays with CCNL2 depletion/overexpression:

• Establish reporter systems for specific splicing events

• Manipulate CCNL2 levels and use antibodies to confirm changes

• Analyze alterations in splicing patterns using RT-PCR or RNA-seq

Proximity ligation assays:

• Detect in situ protein-protein interactions between CCNL2 and splicing factors

• Quantify interaction frequency under different cellular conditions

What is the relationship between CCNL2 and cancer, and how might antibodies help investigate this connection?

Evidence suggests potential connections between CCNL2 and cancer, particularly esophageal squamous cell carcinoma (SCC):

Serum antibody biomarker potential:

• Elevated serum antibody levels against CCNL2 (s-CCNL2-Abs) have been observed in patients with esophageal SCC compared to healthy donors

• These antibodies may serve as potential biomarkers, with a positive rate of approximately 32% in esophageal SCC patients compared to 15% in healthy donors

Expression analysis methodologies:

• Use CCNL2 antibodies for immunohistochemistry to compare expression levels between normal and tumor tissues

• Combine with markers for cell proliferation, apoptosis, and differentiation

• Quantify expression differences using digital pathology tools

Functional studies in cancer models:

• Investigate CCNL2's role in apoptotic pathways using antibodies to track expression and localization changes

• Compare CCNL2 interactions with splicing machinery in normal versus cancer cells

• Examine effects of CCNL2 manipulation on cancer cell phenotypes

p53 pathway connections:

• Research suggests CCNL2 increases the transactivation ability of p53

• This activity appears to be mediated through protein kinase C (PKC), specifically PKCα

• Use co-immunoprecipitation with CCNL2 antibodies to investigate CCNL2-p53 interactions in different cancer contexts

How should I interpret discrepancies between the calculated and observed molecular weights of CCNL2?

The discrepancy between CCNL2's calculated molecular weight (58 kDa) and its observed weight in some Western blot applications (28 kDa) presents an intriguing research question that can be approached methodically:

Experimental approach to resolve molecular weight discrepancies:

• Isoform characterization:

  • Use antibodies targeting different epitopes (N-terminal, C-terminal, internal)

  • Compare banding patterns across antibodies

  • Correlate with known splice variant sequences

• Post-translational modification analysis:

  • Treat samples with phosphatases, deglycosylases, or deubiquitinases

  • Observe shifts in migration patterns

  • Use modification-specific antibodies if available

• Protein processing investigation:

  • Include protease inhibitors during sample preparation

  • Compare fresh vs. stored samples

  • Use pulse-chase experiments to track protein maturation

• Alternative detection methods:

  • Perform mass spectrometry to determine accurate protein mass

  • Use size exclusion chromatography for native molecular weight determination

  • Compare results from different gel systems (Tris-glycine vs. Bis-Tris)

• Recombinant protein comparison:

  • Express tagged full-length CCNL2 and known fragments

  • Run alongside endogenous protein

  • Use as molecular weight standards for accurate comparison

Biological implications:
Understanding this discrepancy may reveal important aspects of CCNL2 biology, including potential regulated proteolytic processing that could have functional significance.

What are common problems when working with CCNL2 antibodies and how can they be resolved?

Issue: Weak or no signal in Western blots

Potential causes and solutions:

• Low expression level: Increase protein loading (50-100 μg); use enrichment strategies

• Inefficient transfer: Optimize transfer conditions for the expected molecular weight

• Antibody concentration too low: Test higher concentrations (1:500 instead of 1:2000)

• Epitope masking: Try different sample preparation methods; consider denaturing conditions

• Wrong molecular weight range: Look at both 58 kDa (calculated) and 28 kDa (observed) regions

Issue: Multiple bands or high background

Potential causes and solutions:

• Non-specific binding: Increase blocking time/concentration; try different blocking agents

• Cross-reactivity: Verify antibody specificity; perform peptide competition assay

• Sample degradation: Add fresh protease inhibitors; keep samples cold

• Secondary antibody issues: Include secondary-only control; try different secondary antibody

Issue: Inconsistent immunoprecipitation efficiency

Potential causes and solutions:

• Insufficient antibody: Increase antibody amount (6-10 μg per reaction)

• Inadequate binding time: Extend incubation to overnight at 4°C

• Buffer incompatibility: Test NETN buffer which has proven effective

• Protein complexes affecting epitope accessibility: Try different antibodies targeting different regions

What emerging research areas could benefit from improved CCNL2 antibody tools?

Single-cell analysis technologies:

• Development of highly specific antibodies suitable for single-cell protein analysis

• Conjugated CCNL2 antibodies for CyTOF or CODEX multiplexed imaging

• Integration with spatial transcriptomics to correlate CCNL2 protein localization with splicing events

Structure-function studies:

• Conformation-specific antibodies that recognize different structural states of CCNL2

• Antibodies that specifically disrupt interactions with certain partners but not others

• Tools to visualize dynamic changes in CCNL2 complexes during splicing reactions

Therapeutic relevance exploration:

• Further investigation of the elevated serum antibody levels against CCNL2 in esophageal SCC patients

• Development of more sensitive detection methods for CCNL2 autoantibodies in patient samples

• Exploration of CCNL2's role in other cancer types where RNA splicing is dysregulated

Combination with emerging technologies:

• CCNL2 antibodies compatible with CRISPR screening approaches

• Integration with proteomics workflows for comprehensive pathway analysis

• Application in organoid or tissue-slice models to study CCNL2 in more physiologically relevant contexts