RIMKLB Antibody

What is RIMKLB and why is it significant in research?

RIMKLB is a protein that catalyzes the synthesis of beta-citryl-L-glutamate and N-acetyl-L-aspartyl-L-glutamate (NAAG). Beta-citryl-L-glutamate is synthesized more efficiently than N-acetyl-L-aspartyl-L-glutamate . RIMKLB belongs to the ATP-grasp family of ligases and produces stoichiometric amounts of NAAG and ADP during its enzymatic reaction .

Alternative names include:

• Beta-citrylglutamate synthase B

• FAM80B

• KIAA1238

• N-acetyl-aspartylglutamate synthetase B (NAAG synthetase B)

• Ribosomal protein S6 modification-like protein B

Understanding RIMKLB is significant for research into neurological systems, as it's predominantly expressed in the CNS and testis , and has emerging relevance in cancer research as indicated by its correlation with immune checkpoint molecules in colorectal cancer .

What types of RIMKLB antibodies are commonly available for research?

Based on the search results, several types of RIMKLB antibodies are available:

Most commercially available RIMKLB antibodies are polyclonal and derived from either rabbit or mouse hosts, designed primarily for Western blotting applications, with some also validated for immunohistochemistry and immunofluorescence.

What are the typical applications for RIMKLB antibodies in experimental research?

RIMKLB antibodies are utilized in several key experimental techniques:

• Western Blotting (WB): The most common application, used for detecting RIMKLB protein expression in tissue or cell lysates. Recommended dilutions typically range from 1:200-1:2000 .

• Immunohistochemistry (IHC): Used to visualize RIMKLB distribution in tissue sections, particularly in testis and CNS tissues. Recommended dilutions are typically 1:50-1:500 .

• Immunofluorescence (IF): Both for paraffin-embedded tissues (IF-P) and cultured cells (ICC), enabling cellular localization studies of RIMKLB. Recommended dilutions are typically 1:50-1:500 .

• ELISA: For quantitative measurement of RIMKLB levels in biological samples .

These applications collectively enable researchers to study RIMKLB expression patterns, subcellular localization, and potential interactions with other proteins across different experimental systems.

How should RIMKLB antibody specificity be validated for research applications?

Validating RIMKLB antibody specificity requires multiple complementary approaches:

• Genetic Validation: Using CRISPR/Cas9 knockout or RNAi knockdown systems to demonstrate reduced or absent antibody signal in samples where RIMKLB expression has been diminished . This is considered the gold standard for antibody validation.

• Orthogonal Validation: Correlating antibody-based detection with antibody-independent methods such as RNA-seq . For example, comparing RIMKLB protein detection in tissues with high vs. low RIMKLB mRNA expression (ideally with at least a 5-fold difference) to confirm correlation.

• Western Blot Analysis: Confirming single-band detection at the expected molecular weight (~42 kDa) for RIMKLB . Multiple bands or unexpected molecular weights may indicate cross-reactivity.

• Testing Across Multiple Cell/Tissue Types: Verifying consistent detection patterns across samples with known RIMKLB expression profiles, especially in CNS and testis tissues where RIMKLB is predominantly expressed .

• Immunogen Sequence Analysis: Evaluating whether the antibody was raised against a unique region of RIMKLB to reduce cross-reactivity with homologous proteins .

As noted by Dr. David Rimm, comprehensive validation should address specificity, sensitivity, and reproducibility to prevent scientific irreproducibility in antibody-based research .

What considerations should guide experimental design when using RIMKLB antibodies?

When designing experiments using RIMKLB antibodies, researchers should consider:

• Appropriate Positive and Negative Controls:

  • Positive controls: Mouse testis tissue for Western blot and IHC; mouse CNS tissue

  • Negative controls: Tissues with confirmed low RIMKLB expression or RIMKLB-knockout samples

• Sample Preparation:

  • For IHC: Antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative

  • For Western blot: Selecting appropriate gel percentage (4-20% Tris-Glycine gradient gels are often used for proteins in RIMKLB's molecular weight range)

• Antibody Dilution Optimization:

  • For Western blot: 1:200-1:1000

  • For IHC/IF: 1:50-1:500

  • Each new antibody lot should be titrated to determine optimal concentrations

• Detection Methods:

  • For Western blot: Consider whether chemiluminescence, fluorescence, or colorimetric detection is most appropriate

  • For IHC/IF: Select appropriate secondary antibodies and visualization systems based on experimental needs

• Quantitative Analysis: Establish appropriate normalization controls when quantifying RIMKLB expression levels

• Experimental Replication: Include biological and technical replicates to ensure reproducibility

How can researchers differentiate between RIMKLA and RIMKLB in experimental systems?

Differentiating between RIMKLA and RIMKLB, which share 65% sequence identity , requires careful experimental planning:

• Antibody Selection: Use antibodies raised against non-homologous regions. The peptide sequences used for RIMKLB-specific antibody production include "CDPESTTEREMLTKLP" and "AGRLTRRMSLLS," which differ from RIMKLA sequences .

• Expression Pattern Analysis: RIMKLA is almost exclusively expressed in the CNS, while RIMKLB is expressed in both CNS and testis . This differential expression pattern can be leveraged to distinguish the proteins.

• Enzymatic Activity Assessment: While both proteins catalyze the synthesis of N-acetylaspartylglutamate, RIMKLB more efficiently catalyzes the synthesis of beta-citryl-L-glutamate . This functional difference can be used to distinguish the two proteins in biochemical assays.

• Molecular Weight Verification: Confirm by Western blot that the detected protein matches the expected molecular weight for RIMKLB (42-48 kDa) .

• Peptide Competition Assays: Use peptides specific to either RIMKLA or RIMKLB in competition assays to demonstrate antibody specificity.

• Gene-Specific Knockdown: Perform selective knockdown of RIMKLA or RIMKLB to confirm antibody specificity.

What is the optimal protocol for Western blotting using RIMKLB antibodies?

Recommended Western Blot Protocol for RIMKLB Detection:

• Sample Preparation:

  • Lyse cells or tissues in RIPA buffer with protease inhibitors

  • Determine protein concentration (BCA or Bradford assay)

  • Prepare samples with loading buffer containing DTT or β-mercaptoethanol

  • Heat samples at 95°C for 5 minutes

• Gel Electrophoresis:

  • Load 20-50 μg protein per lane

  • Use 4-20% Tris-Glycine gradient gels for optimal resolution

  • Run at 100-120V until adequate separation

• Transfer:

  • Transfer to PVDF or nitrocellulose membrane

  • Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking:

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

• Primary Antibody Incubation:

  • Dilute RIMKLB antibody 1:200-1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash 3-5 times with TBST, 5-10 minutes each

• Secondary Antibody Incubation:

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse, depending on primary antibody host)

  • Dilute 1:2000-1:5000 in blocking buffer

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using ECL substrate

  • Expected band size: 42-48 kDa

• Controls:

  • Positive control: Mouse eye tissue or testis lysate

  • Loading control: β-actin, GAPDH, or other housekeeping protein

What are the best practices for immunohistochemistry using RIMKLB antibodies?

Optimal IHC Protocol for RIMKLB Detection:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Deparaffinization and Rehydration:

  • Xylene: 3 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95% ethanol: 2 changes, 3 minutes each

  • 70% ethanol: 1 change, 3 minutes

  • Rinse in distilled water

• Antigen Retrieval:

  • Preferred method: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat in pressure cooker or microwave until boiling, then 10-20 minutes at sub-boiling temperature

• Blocking:

  • Endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Protein block: 5-10% normal serum in PBS for 30 minutes

• Primary Antibody:

  • Dilute RIMKLB antibody 1:50-1:500 in antibody diluent

  • Incubate overnight at 4°C or 1-2 hours at room temperature in a humidified chamber

• Washing:

  • PBS or TBS: 3 changes, 5 minutes each

• Detection System:

  • Apply appropriate HRP-polymer detection system

  • Follow manufacturer's recommendations for incubation times

• Chromogen Development:

  • DAB substrate: Apply for 5-10 minutes or until desired color intensity

  • Monitor microscopically to avoid overdevelopment

• Counterstaining and Mounting:

  • Counterstain with hematoxylin

  • Dehydrate through graded alcohols and xylene

  • Mount with permanent mounting medium

• Controls:

  • Positive tissue control: Mouse testis tissue

  • Negative control: Primary antibody omission or isotype control

How can researchers accurately quantify RIMKLB expression levels in experimental samples?

Accurate quantification of RIMKLB expression requires appropriate methodology selection and rigorous controls:

• Western Blot Quantification:

  • Use appropriate loading controls (β-actin, GAPDH)

  • Ensure signal is within linear range of detection

  • Perform densitometric analysis using software like ImageJ

  • Include a standard curve of recombinant RIMKLB protein for absolute quantification

  • Run at least three biological replicates

• ELISA-Based Quantification:

  • Use validated RIMKLB ELISA kits or develop sandwich ELISA using paired RIMKLB antibodies

  • Include standard curves with recombinant RIMKLB protein

  • Perform technical duplicates or triplicates

  • Ensure samples fall within the linear range of the standard curve

• Immunofluorescence Quantification:

  • Use consistent exposure settings across all samples

  • Employ automated image analysis software with threshold settings

  • Normalize fluorescence intensity to cell number or area

  • Include reference samples of known RIMKLB expression

• RNA-Protein Correlation:

  • Consider parallel qRT-PCR analysis of RIMKLB mRNA levels

  • Correlate protein levels with mRNA expression for orthogonal validation

  • Be aware that post-transcriptional regulation may lead to discrepancies

• Mass Spectrometry Validation:

  • For absolute quantification, consider targeted MS approaches with isotope-labeled standards

  • Can be used to validate antibody-based quantification methods

When publishing quantitative RIMKLB expression data, include complete methodological details including antibody catalog numbers, dilutions, exposure times, and analysis parameters to ensure reproducibility .

Why might researchers encounter non-specific binding with RIMKLB antibodies and how can this be addressed?

Non-specific binding is a common challenge with RIMKLB antibodies. Here are potential causes and solutions:

Causes of Non-Specific Binding:

• Cross-Reactivity with Related Proteins: RIMKLB shares 65% sequence identity with RIMKLA , potentially causing cross-reactivity.

• Suboptimal Blocking: Insufficient blocking allows antibodies to bind non-specifically to the membrane or tissue.

• Excessive Antibody Concentration: Too high primary antibody concentration increases background binding.

• Sample Overloading: Excessive protein can lead to non-specific interactions.

• Degraded Antibody: Antibody quality diminishes over time or with improper storage.

Solutions:

• Antibody Validation and Selection:

  • Use antibodies targeting unique epitopes of RIMKLB

  • Select antibodies validated through genetic approaches (knockout/knockdown)

  • Consider antibodies raised against peptide sequences unique to RIMKLB: "CDPESTTEREMLTKLP" or "AGRLTRRMSLLS"

• Optimization Strategies:

  • Titrate antibody concentrations (try 1:500, 1:1000, 1:2000 dilutions)

  • Test different blocking agents (5% milk, 3-5% BSA)

  • Increase washing duration and frequency

  • Reduce protein loading (20-30 μg may be sufficient)

• Peptide Competition:

  • Pre-incubate the antibody with excess immunogen peptide

  • If the signal disappears, it confirms specificity

• Alternative Detection Methods:

  • Try fluorescent secondary antibodies which may provide cleaner signals

  • Consider more sensitive ECL substrates at lower antibody concentrations

• Sample Preparation:

  • Include phosphatase and protease inhibitors in lysis buffers

  • Consider gentler lysis methods to preserve epitope integrity

What approaches can be taken when RIMKLB antibodies yield inconsistent results between experiments?

Inconsistent results with RIMKLB antibodies may arise from various factors. Here's a systematic approach to address and minimize variability:

Sources of Inconsistency:

• Antibody Lot Variation: Different manufacturing lots may have varying specificities and affinities.

• Sample Preparation Differences: Variations in lysis buffers, fixation times, or antigen retrieval methods.

• Detection System Variability: Inconsistent ECL reagents or developing times.

• Technical Execution: Differences in washing stringency, incubation times, or temperatures.

• Biological Variability: Natural expression differences between biological replicates.

Resolution Strategies:

• Standardize Protocols:

  • Document detailed protocols with exact reagent compositions, incubation times, and temperatures

  • Use consistent sample preparation methods

  • Maintain identical antibody dilutions between experiments

• Antibody Management:

  • Purchase larger antibody lots to ensure consistency across experiments

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

  • Store at recommended temperatures (-20°C for most antibodies)

• Quality Control Measures:

  • Include consistent positive controls in each experiment (e.g., mouse eye tissue or testis for RIMKLB)

  • Run inter-experimental control samples

  • Maintain detailed records of lot numbers and performance

• Validation Across Methods:

  • Confirm findings using alternative detection methods

  • Correlate protein detection with mRNA levels through orthogonal validation

• Statistical Approaches:

  • Increase biological and technical replicates

  • Use appropriate statistical tests to account for experimental variability

  • Consider normalized ratios rather than absolute values when comparing across experiments

• Alternative Antibodies:

  • Test multiple RIMKLB antibodies targeting different epitopes

  • Compare polyclonal vs. monoclonal antibodies for consistency

How can researchers address challenges in detecting low levels of RIMKLB expression?

Detecting low-abundance RIMKLB expression presents technical challenges. Here are specialized approaches to enhance sensitivity:

• Sample Enrichment Techniques:

  • Perform immunoprecipitation to concentrate RIMKLB before detection

  • Use subcellular fractionation to isolate compartments with higher RIMKLB concentration

  • Consider tissue-specific isolation (CNS or testis tissues have higher RIMKLB expression)

• Enhanced Detection Systems:

  • Utilize high-sensitivity ECL substrates for Western blotting

  • Consider tyramide signal amplification (TSA) for IHC/IF to amplify weak signals

  • Use biotin-streptavidin amplification systems

• Optimized Antibody Selection:

  • Choose antibodies with demonstrated high affinity and sensitivity

  • Consider using antibodies targeting the full-length protein (AA 1-307) for maximum epitope availability

• Protocol Modifications:

  • Increase primary antibody incubation time (overnight at 4°C)

  • Reduce washing stringency slightly (shorter wash times or fewer washes)

  • Use more sensitive detection methods (fluorescence over colorimetric)

• Alternative Methodologies:

  • Consider more sensitive techniques like droplet digital PCR for gene expression

  • Employ proximity ligation assay (PLA) for protein detection with single-molecule sensitivity

  • Use mass spectrometry with targeted approaches for RIMKLB peptides

• Control Experiments:

  • Include serial dilutions of positive control samples to establish detection limits

  • Use recombinant RIMKLB as a standard for quantification

  • Include samples with RIMKLB overexpression as positive controls

• Technical Considerations:

  • Ensure fresh antibody dilutions for each experiment

  • Use PVDF membranes instead of nitrocellulose for higher protein binding capacity

  • Minimize protein loss during sample preparation