KHSRP Antibody

What is KHSRP and what cellular functions does it perform?

KHSRP (also known as FUBP2, KSRP, and p75) is a multifunctional RNA-binding protein belonging to the KHSRP family. It performs several critical cellular functions:

• Binds to the dendritic targeting element and may play a role in mRNA trafficking

• Participates in a ternary complex that binds to the downstream control sequence (DCS) of pre-mRNA

• Mediates exon inclusion in transcripts subject to tissue-specific alternative splicing

• May interact with single-stranded DNA from the far-upstream element (FUSE)

• Activates gene expression in certain contexts

• Involved in degradation of inherently unstable mRNAs containing AU-rich elements (AREs) in their 3'-UTR

Recent studies have highlighted KHSRP's role in regulating pre-mRNA splicing by interacting with splicing factor 3b subunit 1 (SF3B1) and enhancing its binding to intronic branch sites .

What types of KHSRP antibodies are available for research?

Several types of KHSRP antibodies are available with different characteristics:

Researchers should select antibodies based on their specific application requirements and target species .

What is the expected molecular weight of KHSRP in Western blot applications?

When performing Western blot analysis, researchers should expect to detect KHSRP at approximately 73-75 kDa:

• Calculated molecular weight: 73 kDa

• Observed molecular weight in most applications: 75 kDa

It's important to note that post-translational modifications, particularly acetylation, may cause slight variations in the observed molecular weight. When validating a new KHSRP antibody, appropriate positive controls (e.g., HEK-293T, HeLa, HepG2, Jurkat, or MCF-7 cells) should be included .

How should KHSRP antibodies be stored and handled for optimal performance?

For maximum stability and performance:

• Store KHSRP antibodies at -20°C

• Most formulations are stable for one year after shipment when properly stored

• Antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations may contain 0.1% BSA for additional stability

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

How can I optimize immunoprecipitation protocols for studying KHSRP interactions?

Optimized immunoprecipitation (IP) protocols for KHSRP typically include:

Standard IP Protocol:

• Harvest cells and lyse with RIPA buffer (150 mM NaCl, 50 mM Tris-HCL, pH 7.4, 1% NP-40, 0.01% SDS, with protease inhibitor cocktail)

• Incubate cell lysates at 4°C overnight with Protein A/G magnetic beads and specific KHSRP antibodies

• Wash immunoprecipitants with lysis buffer three times

• Elute by boiling in SDS sample buffer for SDS-PAGE and immunoblotting

For Co-IP studies of KHSRP interacting partners:

• Prepare cells at >90% confluency

• Scrape cells using immunoprecipitation lysis buffer with protease and phosphatase inhibitors

• Pre-clear 3 mg of protein with 30 μl of protein A/G magnetic beads for 2 hours

• Remove beads and add 12 μl of primary antibody (anti-KHSRP) or isotype IgG to supernatant

• Incubate at 4°C overnight with gentle rocking

• Add 40 μl of protein A/G beads for 4 hours to capture complexes

• Collect beads using a magnetic separator

For recombinant KHSRP antibodies, use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate for optimal results .

What methods are effective for studying KHSRP's role in alternative splicing?

To investigate KHSRP's role in alternative splicing:

• KHSRP Knockdown Approaches:

  • Use small interfering RNA (siKHSRP) in relevant cell lines

  • For in vivo studies, AAV-shKhsrp has been effectively used to reduce KHSRP expression

  • Allow 2-3 weeks post-injection before experimental procedures

• RNA-Sequencing Analysis:

  • Perform whole transcriptome sequencing to identify:

    • Differentially expressed genes (DEGs)

    • KHSRP-regulated alternative splicing events (RASEs)

  • Analysis of MDA-MB-231 cells with KHSRP knockdown identified 1632 DEGs and 1630 RASEs

• Validation of Splicing Changes:

  • Use reverse transcription quantitative PCR (RT-qPCR) to validate changes in splicing patterns

  • Design primers that span exon-exon junctions of interest

  • Focus on functional pathways affected by KHSRP, such as cell cycle and DNA damage/repair pathways

• Interaction Studies with Splicing Factors:

  • Investigate KHSRP interaction with core splicing machinery components such as SF3B1

  • Co-immunoprecipitation followed by Western blotting can confirm these interactions

How can I detect and study post-translational modifications of KHSRP, particularly acetylation?

KHSRP undergoes acetylation, which significantly affects its function. To study this:

• Detection of Acetylated KHSRP:

  • Immunoprecipitate KHSRP from cells treated with deacetylase inhibitors (e.g., 2 μM TSA for 16h and 5 mM NAM for 2h)

  • Perform Western blot with anti-acetyl-lysine antibodies

  • For site-specific detection, use antibodies targeting specific acetylation sites (e.g., KHSRP-acetyl-K205)

• Mass Spectrometric Analysis:

  • Immunoprecipitate KHSRP and perform mass spectrometry to identify acetylation sites

  • Nine lysine residues have been identified as acetylation sites: K-87, -109, -177, -205, -257, -266, -291, -354, and -628

  • K205 is particularly important for KHSRP acetylation

• Site-Directed Mutagenesis:

  • Generate lysine (K) to arginine (R) or glutamine (Q) mutations at specific sites

  • K205R mutation significantly reduces KHSRP acetylation

  • Use KOD-plus-mutagenesis kit or similar systems for generating mutations

• Nuclear/Cytosol Fractionation:

  • Use fractionation kits to separate nuclear and cytosolic components

  • Study the distribution of acetylated KHSRP between cellular compartments

  • Perform immunoprecipitation and Western blotting on fractions with specific antibodies

What are the key considerations when using KHSRP antibodies in cancer research?

KHSRP has been implicated in multiple cancer types. When using KHSRP antibodies in cancer research:

• Selection of Appropriate Cancer Models:

  • KHSRP has been studied in non-small cell lung cancer (NSCLC), prostate cancer (PCa), and breast cancer

  • Different cancer types may show varying KHSRP expression patterns and functions

• Tissue Microarray Analysis:

  • Use IHC on cancer tissue microarrays to evaluate KHSRP expression

  • Apply scoring systems that consider both staining intensity (0-3) and frequency of positive cells (0-4)

  • For acetylation studies, use KHSRP-acetyl-K205 specific antibodies alongside total KHSRP antibodies

• Functional Studies:

  • KHSRP knockdown in MDA-MB-231 breast cancer cells significantly represses proliferation, invasion, and migration while increasing apoptosis

  • In xenograft models, KHSRP acetylation drives tumor growth in prostate cancer

  • Consider pathway analysis focusing on cell cycle, DNA damage repair, cilium assembly, and extracellular matrix organization

• Prognostic Value Assessment:

  • Correlate KHSRP expression with patient survival data

  • Higher KHSRP expression has been associated with worse prognosis in breast cancer patients

What methods should I use to validate KHSRP antibody specificity?

To ensure reliable research results, validate KHSRP antibody specificity using:

• Knockdown/Knockout Controls:

  • Use siRNA, shRNA, or CRISPR-Cas9 to generate KHSRP-depleted samples

  • Compare antibody signal between control and KHSRP-depleted samples

  • Significant reduction in signal confirms antibody specificity

• Recombinant Protein Expression:

  • Express tagged versions of KHSRP (e.g., HA-tagged or Flag-tagged)

  • Perform parallel detection with tag-specific antibodies and KHSRP antibodies

  • Co-localization confirms antibody specificity

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Loss of signal indicates specific binding to target epitope

• Multiple Antibody Comparison:

  • Use antibodies targeting different KHSRP epitopes

  • Consistent results across different antibodies increase confidence in specificity

• Cross-Reactivity Testing:

  • If working with multiple species, test antibody performance in each species

  • Note that reactivity may vary between human, mouse, and rat samples

How can I optimize Western blotting protocols for KHSRP detection?

For optimal Western blot results with KHSRP antibodies:

• Sample Preparation:

  • Use RIPA buffer with protease inhibitors for cell lysis

  • Load 20-40 μg of total protein per lane

  • Positive controls include HEK-293T, HeLa, HepG2, Jurkat, and MCF-7 cells

• Antibody Dilutions:

  • Use dilution ranges of 1:1000-1:6000 for most polyclonal KHSRP antibodies

  • Always optimize dilution for specific antibody and application

• Expected Results:

  • Observe a band at approximately 75 kDa for KHSRP

  • Post-translational modifications may cause slight shifts in apparent molecular weight

What are the methodological approaches for studying KHSRP in animal models?

For studying KHSRP in animal models:

• Viral Vector Delivery:

  • AAV-shKhsrp has been effectively used to knockdown KHSRP in mouse models

  • Allow 2 weeks post-injection before experimental procedures

  • Confirm knockdown efficiency by Western blotting

• Animal Models of Disease:

  • KHSRP has been studied in murine acute liver failure (ALF) models induced by CCl4 or APAP

  • Xenograft models have been used to study KHSRP's role in cancer progression

  • Knockdown of KHSRP exacerbates liver injury in ALF models

• Tissue Analysis:

  • Process tissues for immunohistochemistry, Western blotting, and RNA extraction

  • For brain tissue, both mouse and rat samples have shown detectable KHSRP expression

  • Compare expression patterns between healthy and diseased tissues

How can I investigate KHSRP's role in specific gene regulation?

To study KHSRP's regulatory effects on specific genes:

• RNA Immunoprecipitation (RIP):

  • Use validated KHSRP antibodies (e.g., 55409-1-AP) that are suitable for RIP applications

  • Identify direct RNA targets of KHSRP

• Gene Expression Analysis:

  • After KHSRP knockdown, several genes show altered expression:

    • Downregulated genes in siKHSRP samples: BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1

    • Altered alternative splicing: PARK7, ERCC1, CENPX, and UBE2A

• Pathway Analysis:

  • Focus on pathways enriched in KHSRP-regulated genes:

    • Cell cycle regulation

    • DNA damage and repair

    • Cilium assembly and movement

    • Extracellular matrix organization

What are common issues encountered when using KHSRP antibodies in immunofluorescence?

Challenges in immunofluorescence applications may include:

• High Background:

  • Increase blocking time and concentration

  • Use species-specific serum for blocking

  • Optimize antibody dilution (start with manufacturer recommendations)

• Weak Signal:

  • Increase antibody concentration

  • Extend primary antibody incubation time

  • Use signal amplification systems

  • Ensure appropriate fixation method (paraformaldehyde vs. methanol)

• Non-specific Staining:

  • Pre-adsorb antibody with cell/tissue lysates

  • Include appropriate negative controls (isotype control, KHSRP knockdown cells)

  • Use antibodies validated specifically for IF applications

How do I address inconsistent results in KHSRP detection across different sample types?

When facing inconsistent results:

• Species-Specific Considerations:

  • Confirm antibody reactivity with your species of interest

  • Some antibodies react with human, mouse, and rat samples, while others are human-specific

• Cell/Tissue-Specific Expression:

  • KHSRP expression and localization may vary between tissue types

  • In liver disease models, KHSRP staining is weaker throughout cytoplasm and nuclei compared to untreated controls

• Protocol Optimization:

  • Adjust lysis conditions based on sample type

  • For nuclear proteins, ensure effective nuclear extraction

  • Use phosphatase inhibitors when studying phosphorylated forms of KHSRP

• Antibody Validation:

  • Re-validate antibody with positive and negative controls for each new sample type

  • Consider using multiple antibodies targeting different epitopes