mepce Antibody

What is MEPCE and why is it important in molecular biology research?

MEPCE (Methylphosphate Capping Enzyme) is an S-adenosyl-L-methionine-dependent methyltransferase that adds a methylphosphate cap at the 5'-end of 7SK snRNA, leading to its stabilization . It functions as a component of the 7SK snRNP complex, which plays crucial roles in transcriptional regulation. The importance of MEPCE in research stems from its involvement in RNA processing pathways and its potential implications in cellular processes related to transcription elongation .

In human samples, MEPCE is expressed in chronic myeloid leukemia cells and several normal tissues including adrenal gland, brain, cerebellum, kidney, lung, mammary gland, and testis, with weak or no expression in other tissues . Its subcellular localization is primarily in the nucleus, consistent with its function in RNA processing .

What are the typical applications for MEPCE antibodies in research?

MEPCE antibodies are employed in multiple research techniques, primarily:

The selection of appropriate application depends on research objectives, with Western blot being the most commonly validated method across different antibody products .

What species reactivity should researchers consider when selecting MEPCE antibodies?

Available MEPCE antibodies show reactivity with various species:

Researchers should verify experimental validation for their specific model organism, as some reactivity claims are based on sequence homology rather than empirical testing .

How can researchers optimize MEPCE detection in Western blot applications?

For optimal Western blot detection of MEPCE:

• Sample preparation: MEPCE has a calculated molecular weight of 74.4 kDa but typically migrates at ~90 kDa on SDS-PAGE due to its high proline content (10.7%) . Standard RIPA buffer with protease inhibitors is effective for extraction .

• Gel selection: 8-12% gradient gels provide optimal resolution for the 74-90 kDa range where MEPCE is detected .

• Transfer conditions: Transfer to 0.22 μm nitrocellulose membrane is recommended for optimal protein retention .

• Antibody dilution: Most MEPCE antibodies perform optimally at dilutions between 1:500-1:1,000 for Western blot applications .

• Detection system: Both chemiluminescence and fluorescence-based systems have been successfully employed, with exposure times of approximately 10 seconds reported for strong signals in chemiluminescence .

When analyzing MEPCE cleavage products, particularly in studies involving JMJD6, researchers should note that cleaved forms may appear at ~65 kDa (C-terminal fragment) and ~25 kDa (N-terminal fragment) .

What controls should be included when using MEPCE antibodies in functional studies?

In functional studies investigating MEPCE, several controls are essential:

• Positive controls: Validated cell lines known to express MEPCE including HeLa, MCF-7, and K-562 cells .

• Knockout/knockdown controls: Use of MEPCE knockout or knockdown samples is critical for validating antibody specificity, as demonstrated in studies using Jmjd6 knockout MEF cells as comparative controls .

• Overexpression controls: Overexpression of tagged MEPCE (e.g., His-tagged) provides a useful positive control, especially when studying post-translational modifications or protein interactions .

• Loading controls: Standard loading controls such as actin are recommended for quantitative analyses .

• Antibody validation controls: When possible, use multiple antibodies targeting different epitopes of MEPCE to confirm results, particularly when novel findings are reported .

For experiments investigating MEPCE cleavage by factors like JMJD6, including both wild-type and mutant versions of the interacting protein provides critical validation of specificity .

How should researchers approach epitope selection when studying MEPCE molecular interactions?

The choice of antibody epitope is critical when studying MEPCE interactions and modifications:

• Domain considerations: MEPCE contains distinct functional domains, including the methyltransferase domain. Antibodies targeting different regions may yield different results in interaction studies .

• Post-translational modifications: When studying modifications of MEPCE, avoid antibodies whose epitopes may be affected by the modifications of interest.

• Cleavage studies: In research examining MEPCE cleavage (e.g., by JMJD6), selection of antibodies recognizing either the N-terminal (e.g., His-tag specific) or C-terminal regions is critical. Studies have demonstrated that anti-MePCE antibodies generated against residues 200-250 detect the ~65 kDa C-terminal cleavage product, while N-terminal tag antibodies detect the ~25 kDa N-terminal fragment .

• Available epitope-targeted antibodies:

  • Middle region (various ranges)

  • AA 200-250 region

  • AA 239-267 region

  • AA 639-689 region (C-terminal)

For comprehensive analysis of MEPCE processing, using antibodies targeting different epitopes in parallel provides the most complete characterization .

What are the optimal conditions for MEPCE detection in immunohistochemistry?

For successful immunohistochemical detection of MEPCE:

• Sample preparation: Both formalin-fixed paraffin-embedded (FFPE) and frozen sections have been validated .

• Antigen retrieval:

  • For FFPE tissues, epitope retrieval with citrate buffer pH 6.0 is commonly recommended

  • Alternatively, TE buffer pH 9.0 has been reported as effective for certain antibodies

• Antibody dilutions: Optimal dilutions range from 1:20 to 1:200, with recommendations to titrate for each specific tissue and antibody combination .

• Detection systems: DAB (3,3'-diaminobenzidine) has been successfully employed as a chromogen .

• Positive tissue controls: Human colon cancer tissue has been validated as a positive control for some MEPCE antibodies , while human breast carcinoma has been used for others .

Researchers should note that MEPCE expression patterns vary across tissues, with strongest expression reported in chronic myeloid leukemia cells, adrenal gland, brain, cerebellum, kidney, lung, mammary gland, and testis .

How can dual immunolabeling be optimized for MEPCE co-localization studies?

For co-localization studies involving MEPCE:

• Selection of compatible antibodies: When performing dual labeling, select primary antibodies from different host species (e.g., rabbit anti-MEPCE with mouse anti-partner protein) to avoid cross-reactivity .

• Sequential vs. simultaneous protocols: For nuclear proteins like MEPCE, sequential staining protocols often yield cleaner results than simultaneous incubation with both primary antibodies.

• Fluorophore selection: For immunofluorescence co-localization:

  • Use spectrally distinct fluorophores with minimal overlap

  • For nuclear co-localization, counter-staining with DAPI provides context

  • Consider spectral unmixing for closely related fluorophores

• Recommended partner proteins: Based on MEPCE's functional role, potential co-localization partners include:

  • Other 7SK snRNP components (LARP7, HEXIM1)

  • JMJD6 (for studies of MEPCE processing)

  • RNA Polymerase II components (for transcriptional regulation studies)

• Controls: Include single-stained controls for each antibody to assess bleed-through, and non-immune IgG controls to evaluate non-specific binding .

How should researchers approach immunoprecipitation studies with MEPCE antibodies?

For successful immunoprecipitation of MEPCE and its complexes:

• Antibody selection: Use affinity-purified antibodies specifically validated for IP applications. Several antibodies have been validated for immunoprecipitating MEPCE from human and mouse samples .

• Protocol optimization:

  • Use 2-10 μg antibody per mg of lysate

  • For whole cell lysates, 1 mg lysate for IP has been successfully employed

  • Loading approximately 20% of immunoprecipitated material for Western blot analysis has provided detectable signals

• Buffer considerations: Standard IP buffers containing protease inhibitors are effective for MEPCE immunoprecipitation .

• Detection: For blotting immunoprecipitated MEPCE, antibody concentrations of approximately 1 μg/ml have been effective .

• Co-immunoprecipitation applications: MEPCE antibodies have been successfully used to investigate interactions with components of the 7SK snRNP complex and regulatory proteins like JMJD6 .

For studying dynamic interactions, such as those affected by JMJD6-mediated cleavage, comparative IP experiments using wild-type and knockout/mutant systems provide valuable controls .

What approaches are recommended for investigating MEPCE post-translational modifications?

To study post-translational modifications (PTMs) of MEPCE:

• Proteolytic processing: JMJD6 has been demonstrated to cleave MEPCE both in vivo and in vitro . This cleavage produces:

  • A ~25 kDa N-terminal fragment (detected by N-terminal tag antibodies)

  • A ~65 kDa C-terminal fragment (detected by antibodies targeting residues 200-250)

• Experimental approaches:

  • In vitro cleavage assays using purified components have been successful with recombinant MEPCE (with N-terminal His-tag) and JMJD6 in buffer containing EDTA-free protease inhibitors, α-ketoglutarate, Zn²⁺, and HEPES pH 6.5, incubated at 37°C for 2 hours

  • MALDI-TOF mass spectrometry has been used to analyze MEPCE peptide fragments after proteolytic processing

  • Comparative analysis using wild-type and mutant forms of interacting proteins (e.g., wild-type vs. inactive mutant JMJD6) provides mechanistic insights

• Detection considerations: When analyzing modified forms of MEPCE, note that:

  • Full-length MEPCE has a calculated MW of 74.4 kDa but runs at ~90 kDa on SDS-PAGE due to high proline content

  • The cleaved C-terminal fragment (~65 kDa) is detectable with antibodies targeting residues 200-250

  • Quantification of cleavage products can be performed using ImageJ or similar software

How can researchers address non-specific binding or weak signals when using MEPCE antibodies?

When encountering issues with MEPCE antibody performance:

• Non-specific binding in Western blot:

  • Increase blocking time/concentration (typically 5% non-fat milk or BSA)

  • Optimize primary antibody dilution (1:500-1:5,000 range for Western blot)

  • Increase washing duration and volume

  • Consider using different antibody targeting an alternative epitope

• Weak signals in Western blot:

  • Note that MEPCE is detected at ~90 kDa despite having a theoretical MW of 74.4 kDa due to high proline content

  • Ensure adequate protein loading (20-50 μg total protein)

  • Consider using cell lines with known high MEPCE expression (HeLa, MCF-7, K-562)

  • Decrease antibody dilution (use more concentrated antibody)

  • Increase exposure time for detection

• Issues in immunohistochemistry:

  • Optimize antigen retrieval (try both citrate buffer pH 6.0 and TE buffer pH 9.0)

  • Adjust antibody concentration (1:20-1:200 range)

  • Include positive control tissues (human colon cancer or breast carcinoma)

  • Increase primary antibody incubation time or temperature

• Immunoprecipitation troubleshooting:

  • Increase antibody amount (up to 10 μg/mg lysate)

  • Ensure adequate protein input (≥1 mg lysate)

  • Extend incubation time for antibody-protein binding

  • Consider pre-clearing lysates to reduce non-specific binding

What are the important considerations when interpreting MEPCE cleavage data?

When analyzing MEPCE cleavage, particularly in the context of JMJD6-mediated processing:

• Molecular weight interpretation:

  • Full-length MEPCE: ~90 kDa on SDS-PAGE (theoretical MW: 74.4 kDa)

  • N-terminal cleavage product: ~25 kDa (detected with N-terminal tag antibodies)

  • C-terminal cleavage product: ~65 kDa (detected with antibodies against residues 200-250)

• Antibody selection considerations:

  • For comprehensive analysis, use antibodies targeting different epitopes

  • N-terminal tag antibodies will only detect the N-terminal fragment

  • Antibodies against mid-region or C-terminal epitopes will detect the C-terminal fragment

  • Commercial antibodies with validated epitope regions include those targeting AA 200-250, AA 239-267, and AA 639-689

• Control experiments:

  • Compare wild-type vs. Jmjd6 knockout models

  • Include wild-type vs. inactive mutant JMJD6 overexpression

  • Validate cleavage with both in vivo and in vitro approaches

• Technical artifacts:

  • Distinguish between specific cleavage and non-specific degradation by including appropriate controls

  • Use fresh samples and protease inhibitors to minimize artifactual degradation

  • Include time-course experiments to establish specificity of cleavage events

The detection of cleaved MEPCE forms provides important insights into regulatory mechanisms controlling 7SK snRNP complex function and transcriptional regulation .