QKI Antibody

What is QKI and why is it important in research?

QKI (Quaking) is an RNA-binding protein belonging to the STAR (Signal Transduction and Activation of RNA) family that plays a central role in myelinization. It functions by regulating pre-mRNA splicing, mRNA export, mRNA stability, and protein translation. QKI is expressed in the brain, heart, and other tissues, and is involved in oligodendrocyte differentiation, myelin formation, smooth muscle differentiation, and monocyte to macrophage differentiation . Research interest in QKI has grown due to its associations with schizophrenia, various cancers, and other pathological conditions .

What are the different isoforms of QKI and how do they differ?

QKI has multiple isoforms generated through alternative splicing, with the main variants being:

All isoforms share identical N-terminal structures (amino acids 1-311) containing the KH RNA-binding domain flanked by QUA1 and QUA2 domains, but differ in their C-terminal regions .

How do I select the appropriate QKI antibody for my research?

Selection depends on your experimental goals:

• For detecting all QKI isoforms: Use pan-QKI antibodies targeting the shared N-terminal region (aa 1-311)

• For isoform-specific detection: Use antibodies targeting unique C-terminal regions

  • QKI-5: Antibodies targeting the nuclear localization signal (aa 315-331, ATKVRRHDMRVHPYQRI)

  • QKI-7: Antibodies targeting the unique C-terminal tail (aa 303-317, LEWIEMPVMPDISAH)

• Consider application compatibility (WB, IHC, IF, ICC) and host species to avoid cross-reactivity issues

• Verify species reactivity matches your experimental model (human, mouse, rat)

What are the optimal conditions for Western blotting with QKI antibodies?

For optimal Western blotting results with QKI antibodies:

• Sample preparation:

  • Use 20-30 μg of total protein from lysates

  • Brain tissue lysates work well as positive controls

• Recommended dilutions:

  • Pan-QKI antibodies: 1:1000 to 1:3000

  • Isoform-specific antibodies: 1:1000

• Expected bands:

  • QKI-5: ~38-42 kDa

  • QKI-6/7/7b: ~36-38 kDa

• Signal development:

  • For colorimetric detection: 1 μg/ml of antibody is sufficient with appropriate secondary antibodies (e.g., Goat anti-mouse IgG:HRP)

  • For enhanced sensitivity: Consider using ECL-based detection systems

• Validation controls:

  • Positive controls: Brain lysates, Neuro-2a cells, PC-12 cells

  • Negative controls: QKI knockout/knockdown samples

How can I optimize immunohistochemistry protocols with QKI antibodies?

For successful immunohistochemistry with QKI antibodies:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) or frozen sections can be used

  • For FFPE sections, antigen retrieval is critical

• Antigen retrieval methods:

  • TE buffer pH 9.0 (recommended)

  • Alternative: Citrate buffer pH 6.0

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from the species of the secondary antibody

  • Primary antibody dilutions: 1:100 to 1:600 depending on the specific antibody

  • Incubate overnight at 4°C for optimal results

• Detection systems:

  • HRP/DAB-based detection works well for chromogenic visualization

  • For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies

• Controls:

  • Positive control tissues: Brain, stomach tissue

  • Negative controls: Primary antibody omission and QKI-deficient tissues

What are the considerations for immunofluorescence applications with QKI antibodies?

For immunofluorescence applications:

• Cell fixation and permeabilization:

  • 4% paraformaldehyde for 10-15 minutes for fixation

  • 0.1-0.3% Triton X-100 for permeabilization

• Antibody dilutions:

  • Primary antibody: 1:100 to 1:400 for most QKI antibodies

  • For conjugated antibodies (e.g., Alexa Fluor 488-conjugated), 1:100 to 1:200

• Expected cellular localization:

  • QKI-5: Primarily nuclear

  • QKI-6/7/7b: Primarily cytoplasmic

• Co-staining considerations:

  • Compatible with other antibodies for co-localization studies

  • Consider spectral overlap when selecting fluorophores

• Imaging parameters:

  • Confocal microscopy provides better resolution for subcellular localization studies

  • Use appropriate exposure settings to avoid photobleaching

How can I troubleshoot weak or absent signals in Western blots with QKI antibodies?

When experiencing weak or absent signals:

• Protein extraction optimization:

  • Use RIPA buffer with protease inhibitors

  • Ensure complete lysis and proper protein quantification

• Antibody-related factors:

  • Increase antibody concentration (try 1:500 if 1:1000 doesn't work)

  • Extend incubation time (overnight at 4°C)

  • Check antibody stability and storage conditions (aliquot and store at -20°C)

• Detection system factors:

  • Use more sensitive ECL reagents

  • Increase exposure time

  • Consider fresh secondary antibodies

• Sample-related factors:

  • QKI expression may vary by tissue/cell type

  • Verify QKI expression in your sample using published literature

  • Use positive controls (brain tissue) alongside your samples

• Technical considerations:

  • Ensure proper transfer of proteins to membrane

  • Optimize blocking conditions (duration and blocking agent)

  • Verify that antibody recognizes native/denatured protein as appropriate

How can I validate the specificity of QKI antibodies in my experimental system?

To validate antibody specificity:

• Genetic approaches:

  • Use QKI knockout/knockdown samples as negative controls

  • Compare wild-type vs. QKI-deficient samples

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • This should abolish specific binding

• Multiple antibody validation:

  • Use multiple antibodies targeting different epitopes

  • Compare staining patterns between pan-QKI and isoform-specific antibodies

• Recombinant protein controls:

  • Use purified QKI proteins as positive controls

  • Verify band sizes match expected molecular weights

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that the immunoprecipitated protein is indeed QKI

  • This approach identifies potential cross-reactive proteins

How can QKI antibodies be used to study differential expression of QKI isoforms in pathological conditions?

To study differential expression of QKI isoforms:

• Comparative analysis methodology:

  • Use isoform-specific antibodies (QKI-5, QKI-7) alongside pan-QKI antibodies

  • Quantify relative expression levels by densitometry

• Cellular localization analysis:

  • Perform subcellular fractionation (nuclear vs. cytoplasmic)

  • Use immunofluorescence to visualize distribution patterns

• Disease model applications:

  • Compare control vs. disease tissues (e.g., schizophrenia, myelination disorders)

  • Look for changes in isoform ratios rather than just total QKI levels

• Temporal expression analysis:

  • Track expression changes during development or disease progression

  • Correlate with functional outcomes (e.g., myelination status)

• Multi-omics integration:

  • Combine protein data with RNA-seq to assess transcriptional vs. post-transcriptional regulation

  • Correlate with functional readouts of QKI activity

How can QKI antibodies be employed in RNA-protein interaction studies?

For RNA-protein interaction studies:

• RNA immunoprecipitation (RIP):

  • Use QKI antibodies to pull down QKI-RNA complexes

  • Recommended dilution: 1:100 for IP applications

  • Follow with qRT-PCR or sequencing to identify bound RNAs

• Cross-linking immunoprecipitation (CLIP):

  • UV cross-linking to stabilize RNA-protein interactions

  • Use QKI antibodies optimized for eCLIP applications (1:200 dilution)

  • Combine with high-throughput sequencing to map binding sites

• Proximity ligation assays:

  • Detect in situ interactions between QKI and other RNA-binding proteins

  • Requires co-optimization of QKI antibodies with antibodies against potential partners

• Immunofluorescence colocalization:

  • Combine QKI antibodies with RNA FISH

  • Visualize spatial relationships between QKI and target RNAs

• In vitro binding validation:

  • Use recombinant QKI and synthetic RNAs

  • Confirm antibody recognition of protein-RNA complexes

What are the considerations for using QKI antibodies in studying post-translational modifications?

For studying QKI post-translational modifications:

• Phosphorylation analysis:

  • QKI contains tyrosine clusters within proline-rich PXXP motifs that can be phosphorylated by Src kinases

  • Use phospho-specific antibodies alongside total QKI antibodies

  • Treat samples with phosphatase inhibitors during extraction

• Detection approaches:

  • Immunoprecipitate with QKI antibodies, then probe with phospho-tyrosine antibodies

  • Use PhosTag gels to separate phosphorylated from non-phosphorylated forms

• Functional correlation:

  • Compare modification status with RNA-binding activity

  • Assess how modifications alter subcellular localization

• Stimulus response studies:

  • Track changes in modification status following relevant stimuli

  • Correlate with functional outcomes

• Mass spectrometry validation:

  • Immunoprecipitate QKI and analyze by mass spectrometry

  • Identify and quantify specific modification sites

How should I interpret differences in QKI isoform expression patterns across tissues and experimental conditions?

For interpreting QKI isoform expression patterns:

• Tissue-specific considerations:

  • Brain: All QKI isoforms are expressed, with QKI-5 predominant in oligodendrocytes

  • Heart: QKI shows differential expression during development and pathological conditions

  • Other tissues: Expression patterns may vary and should be established experimentally

• Isoform ratio analysis:

  • Calculate relative ratios between isoforms

  • Compare these ratios across conditions rather than absolute values

  • Consider using quantitative Western blotting with standard curves

• Functional correlation:

  • QKI-5 (nuclear): Changes may indicate altered splicing regulation

  • QKI-6/7 (cytoplasmic): Changes may reflect altered mRNA stability or translation

• Methodological considerations:

  • Confirm protein-level changes with mRNA analysis

  • Use multiple antibodies to validate observations

  • Include appropriate biological replicates and statistical analysis

• Publication standards:

  • Report antibody catalog numbers, dilutions, and detection methods

  • Include supporting validation data (specificity controls)

  • Document quantification methods and statistical approaches

How can I design experiments to study QKI's role in specific RNA processing events using QKI antibodies?

To design experiments for studying QKI's role in RNA processing:

• Experimental workflow design:

  • Establish baseline QKI expression in your model system

  • Manipulate QKI levels (overexpression, knockdown)

  • Assess consequences on target RNA processing

  • Use QKI antibodies to confirm manipulation success

• RNA splicing analysis:

  • Use QKI-5 specific antibodies for studying nuclear splicing events

  • Combine with RT-PCR for specific splice variants

  • RIP-seq to identify directly bound pre-mRNAs

• mRNA stability studies:

  • Use pan-QKI or cytoplasmic isoform-specific antibodies

  • Actinomycin D chase experiments to measure half-life of target mRNAs

  • Correlate QKI binding with stability changes

• Integrated approaches:

  • Combine protein studies (using antibodies) with transcriptomic analyses

  • CLIP-seq to identify direct binding targets

  • Manipulate binding sites and assess functional consequences

• Disease model applications:

  • Compare RNA processing events between normal and disease states

  • Correlate with QKI isoform expression patterns

  • Rescue experiments to establish causality

What are the key specifications of commonly used QKI antibodies that researchers should be aware of?

Below is a comparative table of commonly used QKI antibodies: