LOH12CR1 Antibody

What is LOH12CR1 and why is it significant for research?

LOH12CR1 (loss of heterozygosity, 12, chromosomal region 1), also known as BORCS5, is part of the BORC complex involved in lysosome movement and localization at the cell periphery. It may indirectly play a role in cell spreading and motility . Recent research has identified LOH12CR1 as a potential tumor suppressor gene, particularly in colorectal cancer, where it has been shown to inhibit cancer cell proliferation by regulating the G1/S cell cycle transition through p16 INK4a and p21 WAF1/CIP1 pathways . The gene is located within the 12p12-13 region, an area where loss of heterozygosity has been frequently observed in various cancer types .

What are the key considerations when selecting a LOH12CR1 antibody for research?

When selecting a LOH12CR1 antibody, researchers should consider:

• Target specificity: Verify that the antibody specifically recognizes LOH12CR1 and has minimal cross-reactivity with other proteins

• Reactivity with species of interest: Different antibodies show reactivity with human, mouse, or rat samples

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF/ICC, ELISA)

• Clonality: Choose between polyclonal and monoclonal options based on your research needs:

  • Polyclonal antibodies typically offer broader epitope recognition

  • Monoclonal antibodies provide more consistent results between experiments

• Antibody format: Consider whether you need conjugated antibodies (e.g., HRP-conjugated , DyLight 680-conjugated ) or unconjugated antibodies

What are the known isoforms of LOH12CR1 and how do they affect antibody selection?

LOH12CR1 has two identified isoforms with molecular weights of approximately 17 kDa and 22 kDa . When selecting an antibody, it's important to consider which isoform(s) you wish to detect. Most commercially available antibodies detect both isoforms, with the observed molecular weight typically falling in the 17-22 kDa range . Some antibodies target specific regions of the protein:

• C-terminal targeted antibodies (e.g., amino acids 167-196)

• Middle region targeted antibodies (e.g., amino acids 53-123)

The choice depends on your research question and whether a specific isoform is relevant to your study. Review the validation data from suppliers to confirm which isoform(s) the antibody detects.

What are the recommended protocols for Western Blot detection of LOH12CR1?

For optimal Western Blot detection of LOH12CR1:

Sample Preparation:

• Prepare cell/tissue lysates in RIPA buffer with protease inhibitors

• Load 20-30 μg of total protein per lane

• Use fresh samples when possible as LOH12CR1 may be susceptible to degradation

Recommended Protocol:

• Separate proteins on a 12-15% SDS-PAGE gel (optimal for lower molecular weight proteins)

• Transfer to PVDF membrane (preferred over nitrocellulose for small proteins)

• Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary antibody at recommended dilution (typically 1:500-1:2000) overnight at 4°C

• Wash 3x with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody

• Wash 3x with TBST, 5 minutes each

• Detect signal using ECL substrate

Expected Results:

• Look for bands between 17-22 kDa

• Positive controls: HeLa cells, Jurkat cells, and K-562 cells have shown reliable expression

How should immunohistochemistry be optimized for LOH12CR1 detection in cancer tissues?

For effective immunohistochemical detection of LOH12CR1 in cancer tissues:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Embed in paraffin and section at 4-5 μm thickness

• Mount on positively charged slides

Antigen Retrieval Options:

• Primary recommendation: TE buffer at pH 9.0

• Alternative: Citrate buffer at pH 6.0

Staining Protocol:

• Deparaffinize and rehydrate sections

• Perform antigen retrieval

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with 5% normal serum

• Incubate with primary antibody at dilution 1:50-1:500 (optimize for your specific antibody)

• Incubate with appropriate detection system (e.g., polymer-based)

• Develop with DAB and counterstain with hematoxylin

Validation Controls:

• Positive tissue controls: Human lung cancer and liver cancer tissues have shown reliable expression

• Consider using LOH12CR1 recombinant protein for blocking experiments to confirm specificity

What methodologies are recommended for studying LOH12CR1's function in cancer cell models?

Based on published research, the following methodologies are effective for studying LOH12CR1's function in cancer:

1. Gene Expression Manipulation:

• Knockdown: siRNA or shRNA targeting LOH12CR1 (shown to promote colorectal cancer cell proliferation and colony formation)

• Overexpression: Transfection with LOH12CR1-expressing vectors (shown to inhibit proliferation)

2. Functional Assays:

• Cell proliferation assays (MTT, BrdU incorporation)

• Colony formation assays

• Cell cycle analysis by flow cytometry (particularly focusing on G1/S transition)

• Migration and invasion assays (given LOH12CR1's potential role in cell motility)

3. Pathway Analysis:

• Western blot analysis of p16 INK4a and p21 WAF1/CIP1 expression levels

• Co-immunoprecipitation to identify protein-protein interactions

• Lysosomal localization studies using co-staining with lysosomal markers

4. Clinical Correlation:

• Immunohistochemical analysis of patient samples with correlation to clinical outcomes

• Comparison of expression levels between tumor and adjacent normal tissues

A comprehensive study by researchers found that LOH12CR1 knockdown accelerated G1/S cell cycle transition through downregulation of p16 INK4a and p21 WAF1/CIP1, while ectopic expression showed opposite effects .

How can LOH12CR1 antibodies be used to investigate its tumor suppressor role in different cancer types?

LOH12CR1 has been identified as a potential tumor suppressor in colorectal cancer , but its role in other cancer types warrants investigation. Researchers can:

1. Conduct Comparative Expression Analysis:

• Use immunohistochemistry with LOH12CR1 antibodies across tissue microarrays representing multiple cancer types

• Compare expression levels between tumor and matched normal tissues

• Correlate expression with clinical parameters (stage, grade, survival)

2. Investigate Molecular Mechanisms:

• Examine whether the p16/p21 regulatory pathway identified in colorectal cancer is conserved across cancer types

• Study LOH12CR1's interaction with the BORC complex in different cancer cell lines

• Investigate whether lysosomal positioning (regulated by LOH12CR1) affects cancer cell behavior differently across cancer types

3. Genetic Analysis:

• Analyze the 12p12-13 region for loss of heterozygosity across cancer types

• Correlate LOH status with LOH12CR1 protein expression using antibody-based detection

• Perform mutation analysis of LOH12CR1 in tumors with low protein expression

4. Therapeutic Implications:

• Test whether restoring LOH12CR1 expression sensitizes cancer cells to specific therapies

• Develop screening assays using LOH12CR1 antibodies to identify compounds that upregulate its expression

A study of 174 colorectal cancer tissues showed significantly decreased LOH12CR1 protein in cancerous tissues compared to normal tissues (p<0.001), with expression levels negatively correlating with clinical prognosis . This methodology can be extended to other cancer types.

What are the best approaches for studying the interaction between LOH12CR1 and the BORC complex using antibody-based techniques?

To investigate LOH12CR1 (BORCS5) interactions with other BORC complex components:

1. Co-immunoprecipitation (Co-IP):

• Immunoprecipitate with anti-LOH12CR1 antibody

• Analyze precipitates for other BORC components by Western blot

• Reverse Co-IP using antibodies against other BORC components to confirm interactions

• Consider using crosslinking agents to stabilize transient interactions

2. Proximity Ligation Assay (PLA):

• Use LOH12CR1 antibody paired with antibodies against other BORC components

• This technique allows visualization of protein-protein interactions in situ

• Quantify interaction signals in different cellular contexts

3. Immunofluorescence Co-localization:

• Perform double immunofluorescence with LOH12CR1 antibody and antibodies against:

  • Other BORC components

  • Lysosomal markers

  • Microtubule or cytoskeletal proteins

• Use super-resolution microscopy for detailed co-localization analysis

4. FRET (Förster Resonance Energy Transfer):

• Use fluorophore-conjugated LOH12CR1 antibodies (such as DyLight 680-conjugated )

• Pair with differently labeled antibodies against other BORC components

• Analyze energy transfer to determine close proximity of proteins

When conducting these experiments, it's essential to validate antibody specificity using appropriate controls, including recombinant LOH12CR1 protein or cells with LOH12CR1 knockdown/knockout.

How can quantitative analysis of LOH12CR1 expression be correlated with cancer patient outcomes?

For rigorous correlation between LOH12CR1 expression and cancer outcomes:

1. Tissue Microarray (TMA) Analysis:

• Create TMAs from patient cohorts with complete clinical follow-up

• Stain with validated LOH12CR1 antibody using standardized IHC protocol

• Develop scoring system:

  • H-score (intensity × percentage of positive cells)

  • Digital image analysis for objective quantification

2. Statistical Analysis Workflow:

• Determine optimal cutoff values for "high" vs. "low" expression

• Perform Kaplan-Meier survival analysis

• Use Cox proportional hazards model for multivariate analysis

• Correlate with established prognostic markers

3. Multi-marker Analysis:

• Combine LOH12CR1 with p16 INK4a and p21 WAF1/CIP1 expression analysis

• The protein level of LOH12CR1 has been shown to correlate with expression of p16 INK4a and p21 WAF1/CIP1

• Create prognostic models incorporating multiple markers

4. Longitudinal Analysis:

• Analyze samples from different disease stages

• Compare primary tumors with metastatic lesions

• Evaluate changes in expression during therapy

In a study of colorectal carcinomas, LOH12CR1 protein levels negatively correlated with clinical prognosis, suggesting its potential value as a prognostic biomarker . Similar approaches can be applied to other cancer types to determine if this correlation is broadly applicable.

What are common challenges when working with LOH12CR1 antibodies and how can they be addressed?

Common challenges and solutions when working with LOH12CR1 antibodies include:

1. Weak or No Signal:

• Increase antibody concentration gradually

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

• Extend primary antibody incubation time or temperature

• For Western blot, ensure transfer efficiency for small proteins is optimal

• Fresh lysates may be required as LOH12CR1 could be degradation-sensitive

2. High Background:

• Optimize blocking conditions (try both milk and BSA)

• Increase washing steps duration and number

• Decrease antibody concentration

• Pre-absorb the antibody with the blocking agent

• For IHC, optimize endogenous peroxidase blocking

3. Non-specific Bands in Western Blot:

• Verify protein loading amount (20-30 μg recommended)

• Increase gel percentage (15% may be optimal for small proteins)

• Use positive controls (HeLa, Jurkat, K-562 cells)

• Perform validation with LOH12CR1 recombinant protein

4. Variable Results Between Experiments:

• Standardize protocols rigorously

• Use the same lot of antibody when possible

• Include positive and negative controls in each experiment

• Consider using monoclonal antibodies for more consistent results

How can researchers validate the specificity of LOH12CR1 antibodies for their experimental systems?

To validate LOH12CR1 antibody specificity:

1. Blocking Experiments:

• Pre-incubate antibody with LOH12CR1 recombinant protein control fragment

• For example, use 100x molar excess of the protein fragment based on antibody concentration

• Pre-incubate for 30 minutes at room temperature before application

• Signal elimination confirms specificity

2. Genetic Validation:

• Test antibody in cells with LOH12CR1 knockdown or knockout

• Compare signal in wild-type vs. modified cells

• Signal reduction/elimination confirms specificity

3. Multiple Antibody Validation:

• Test multiple antibodies targeting different epitopes of LOH12CR1

• Compare staining/detection patterns

• Consistent results increase confidence in specificity

4. Mass Spectrometry Validation:

• Immunoprecipitate with LOH12CR1 antibody

• Analyze precipitated proteins by mass spectrometry

• Confirm presence of LOH12CR1 and expected interacting partners

5. Tissue/Cell Type Controls:

• Use tissues/cells with known LOH12CR1 expression patterns

• Positive controls: HeLa, Jurkat, K-562 cells for WB ; human lung and liver cancer tissues for IHC

• U2OS cells for immunofluorescence

What are the recommended dilution ratios for different experimental applications of LOH12CR1 antibodies?

Based on the search results, here are the recommended dilution ratios for different applications of LOH12CR1 antibodies:

Important considerations:

• These are starting points; optimization is necessary for each experimental system

• Sample-dependent factors may require adjustment of dilutions

• For conjugated antibodies (e.g., HRP-conjugated, DyLight 680-conjugated), follow specific recommendations for the conjugate

• When using blocking peptides, a 100x molar excess of the protein fragment based on antibody concentration is recommended

• It is advised to titrate antibodies in each testing system to obtain optimal results

Always validate these dilutions in your specific experimental system with appropriate positive and negative controls.