MKLN1 Antibody, Biotin conjugated

What is MKLN1 protein and what cellular functions is it involved in?

MKLN1 (muskelin 1) is an intracellular protein that acts as a mediator of cell spreading and cytoskeletal responses to the extracellular matrix component THBS1 (thrombospondin-1). The protein has a molecular weight of approximately 60,739 Da and is also known by the gene alias TWA2 . Recent research has revealed that MKLN1 is a substrate of the CTLH Ubiquitin Ligase complex and forms a complex with ZMYND19 that negatively regulates mTOR signaling . This involvement in mTOR regulation suggests MKLN1 plays a role in cellular processes related to growth, metabolism, and stress response. The protein exhibits a half-life of approximately 7.7 hours in control cells, but is significantly stabilized when components of the CTLH complex are knocked out .

What are the key specifications of MKLN1 Antibody, Biotin conjugated?

The MKLN1 Antibody, Biotin conjugated is a rabbit polyclonal antibody developed for specific detection of human MKLN1 protein. Its key technical specifications include:

This antibody has been purified using Protein G affinity chromatography, ensuring high specificity and low background in experimental applications .

What detection methods are compatible with this biotinylated antibody?

The biotin conjugation of this MKLN1 antibody makes it particularly versatile for multiple detection systems. The antibody has been shown to work effectively in applications such as Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme Immunoassay (EIA), and other immunoassay formats . The biotin-streptavidin system offers one of the strongest non-covalent biological interactions known, with a dissociation constant (Kd) of approximately 10^-15 M, making it an excellent choice for sensitive detection methods.

For detection, researchers can employ streptavidin conjugated to various reporter molecules like horseradish peroxidase (HRP), alkaline phosphatase, fluorophores, or quantum dots. This versatility allows for colorimetric, chemiluminescent, or fluorescent visualization depending on the experimental requirements. The streptavidin-biotin interaction can also be leveraged for protein isolation techniques, similar to those described for cell surface biotinylated proteins, where biotinylated proteins are adsorbed onto streptavidin-agarose for purification .

How should I optimize ELISA protocols when using MKLN1 Antibody, Biotin conjugated?

When optimizing ELISA protocols with MKLN1 Antibody, Biotin conjugated, several methodological considerations can enhance sensitivity and specificity:

• Antibody titration: Begin with a checkerboard titration to determine the optimal concentration of the biotinylated antibody. Typically starting with dilutions ranging from 1:500 to 1:5000 is recommended.

• Blocking optimization: Use 2-5% BSA or casein in PBS-T (PBS with 0.05% Tween-20) as blocking buffer to minimize non-specific binding. The blocking step should be performed for at least 1 hour at room temperature or overnight at 4°C.

• Detection system selection: Utilize streptavidin-HRP for colorimetric assays with substrates like TMB (3,3',5,5'-tetramethylbenzidine) or streptavidin-fluorophore conjugates for fluorescence-based detection.

• Signal amplification: For enhanced sensitivity, consider incorporating a tyramide signal amplification (TSA) system with the biotinylated antibody, which can improve detection by 10-100 fold.

• Washing steps: Implement stringent washing (4-5 washes with PBS-T) between each step to reduce background while preserving specific signal.

Each of these parameters should be systematically tested to establish the optimal protocol for your specific experimental conditions and sample types .

What are the best practices for storage and handling to maintain antibody activity?

The biotinylated MKLN1 antibody requires specific storage and handling conditions to maintain its activity and specificity over time:

• Storage temperature: Store the antibody at -20°C for long-term preservation. For short-term use (up to one week), it may be kept at 2-8°C .

• Aliquoting: Upon receipt, divide the stock solution into small aliquots to prevent repeated freeze-thaw cycles. Each freeze-thaw cycle can reduce antibody activity by approximately 10-15%.

• Buffer considerations: The antibody is typically supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose as stabilizers. Avoid diluting the stock solution unless immediately before use .

• Contamination prevention: Use sterile technique when handling the antibody to prevent microbial contamination.

• Sodium azide precautions: Note that the preservative sodium azide is poisonous and hazardous, requiring proper handling by trained personnel .

• Freeze-thaw management: If multiple uses are necessary, rapidly thaw the antibody at 37°C and then place it on ice. Return unused portions to -20°C as quickly as possible to minimize degradation.

Following these guidelines will help ensure consistent antibody performance across experiments and maximize shelf-life .

How can I use MKLN1 Antibody, Biotin conjugated to investigate MKLN1's role in the CTLH ubiquitin ligase pathway?

The CTLH (C-terminal to LisH) ubiquitin ligase complex regulates the stability of MKLN1, and investigating this relationship requires specialized methodological approaches:

• Co-immunoprecipitation studies: Use the biotinylated MKLN1 antibody with streptavidin beads to pull down MKLN1-associated proteins. Subsequently analyze the precipitates for CTLH complex components (MAEA, RANBP9, etc.) using Western blotting or mass spectrometry.

• Protein stability assays: Employ cycloheximide chase experiments in conjunction with MKLN1 detection using the biotinylated antibody to track protein degradation rates. This approach can reveal how MKLN1 stability is altered in cells with CTLH components knocked down or overexpressed .

• Ubiquitination detection: Combine immunoprecipitation of MKLN1 using the biotinylated antibody with ubiquitin detection to directly visualize MKLN1 ubiquitination status under various conditions.

• Functional rescue experiments: In CTLH-perturbed cells where MKLN1 is stabilized, use siRNA against MKLN1 together with downstream functional assays to determine the consequences of MKLN1 accumulation. The biotinylated antibody can be used to confirm knockdown efficiency.

Recent research has demonstrated that MKLN1 is among the proteins most significantly increased by CTLH perturbation, with its half-life extending beyond 7.7 hours when CTLH components are knocked out . This makes MKLN1 detection critical for understanding CTLH-mediated protein quality control mechanisms.

What approaches can I use to study the interaction between MKLN1 and ZMYND19 in mTOR regulation?

Recent research has uncovered a critical role for MKLN1 and ZMYND19 in negatively regulating mTOR signaling. To study this interaction using the biotinylated MKLN1 antibody:

• Proximity ligation assays (PLA): Combine the biotinylated MKLN1 antibody with a primary antibody against ZMYND19, followed by appropriate secondary antibodies with PLA probes to visualize protein-protein interactions in situ with subcellular resolution.

• FRET-based interaction studies: Use the biotinylated MKLN1 antibody with streptavidin-conjugated fluorophores as FRET donors, and anti-ZMYND19 antibodies with compatible fluorophores as FRET acceptors to measure interaction dynamics in live or fixed cells.

• Co-localization analysis: Employ immunofluorescence microscopy with the biotinylated MKLN1 antibody (detected with streptavidin-fluorophore) and ZMYND19 antibody to examine spatial co-localization, particularly at lysosomal membranes where mTORC1 is activated.

• Sequential immunoprecipitation: Perform tandem immunoprecipitation, first with MKLN1 antibody followed by ZMYND19 antibody (or vice versa), to isolate the pure MKLN1-ZMYND19 complex for functional studies.

Research has shown that MKLN1 depletion increases steady-state ZMYND19 levels, suggesting that MKLN1 supports ZMYND19 turnover . Additionally, double knockout studies of ZMYND19/MKLN1 revealed significant rescue of alpelisib-treated cells with MAEA knockout, pointing to a partially redundant or joint role in cell survival regulation .

How do I design experiments to assess MKLN1's impact on cytoskeletal dynamics using the biotinylated antibody?

MKLN1 is known to mediate cell spreading and cytoskeletal responses to the extracellular matrix component thrombospondin-1 (THBS1). To investigate these functions:

• Live-cell imaging: Combine the biotinylated MKLN1 antibody with cell-permeable streptavidin-fluorophore conjugates for dynamic tracking of MKLN1 redistribution during cytoskeletal rearrangements.

• Co-sedimentation assays: Use the biotinylated antibody to detect MKLN1 in actin co-sedimentation fractions to quantify its association with the cytoskeleton under various conditions (e.g., THBS1 stimulation, cytoskeletal drug treatments).

• Cell spreading analysis: Perform time-course immunostaining after plating cells on THBS1-coated surfaces, using the biotinylated MKLN1 antibody to correlate MKLN1 localization with focal adhesion formation and actin reorganization.

• FRAP (Fluorescence Recovery After Photobleaching): Use the biotinylated antibody with fluorescent streptavidin to perform FRAP analyses at cytoskeletal structures, determining MKLN1 mobility and exchange rates at these sites.

• Super-resolution microscopy: Implement techniques like STORM or PALM with the biotinylated antibody to achieve nanoscale resolution of MKLN1 distribution relative to cytoskeletal components.

These approaches allow for comprehensive analysis of MKLN1's dynamic role in mediating cellular responses to extracellular matrix components .

What are common issues when using MKLN1 Antibody, Biotin conjugated and how can they be resolved?

When working with biotinylated MKLN1 antibody, researchers may encounter several challenges:

• High background signal: This commonly results from insufficient blocking or excessive antibody concentration. Solution: Optimize blocking conditions using 5% BSA or 5% non-fat milk in PBS-T and titrate the antibody concentration. Additionally, pre-absorb the antibody with cell/tissue lysate not expressing the target protein.

• Poor signal strength: May be caused by low target protein expression or antibody degradation. Solution: Increase sample concentration, enhance signal with amplification systems like TSA (tyramide signal amplification), or use fresh antibody aliquots. Verify adequate biotin conjugation using streptavidin-based detection.

• Non-specific bands in Western blotting: Could indicate cross-reactivity with similar epitopes. Solution: Increase washing stringency, optimize antibody dilution, and include appropriate negative controls. Use peptide competition assays to confirm specificity.

• Inconsistent results between experiments: Often stems from variations in antibody handling. Solution: Standardize protocols, avoid repeated freeze-thaw cycles by preparing single-use aliquots, and ensure consistent incubation times and temperatures.

• Interference from endogenous biotin: Particularly problematic in biotin-rich tissues like liver or kidney. Solution: Include an avidin/streptavidin blocking step prior to applying the biotinylated antibody to mask endogenous biotin.

Each of these challenges can be systematically addressed through careful optimization and control experiments .

How can I compare the performance of MKLN1 Antibody, Biotin conjugated versus unconjugated versions?

To effectively compare the performance of biotinylated versus unconjugated MKLN1 antibodies:

• Parallel titration: Run side-by-side titration curves using identical samples and standardized protocols, evaluating signal-to-noise ratios at each dilution to determine optimal working concentrations for each format.

• Sensitivity comparison: Prepare serial dilutions of purified MKLN1 protein or cell lysates with known MKLN1 expression levels to determine the lower limit of detection for each antibody format. The detection system should be equivalent (e.g., streptavidin-HRP for biotinylated antibody, matched secondary-HRP for unconjugated).

• Specificity assessment: Perform Western blots with both antibody formats on lysates from multiple cell types, including MKLN1 knockout or knockdown samples as negative controls. Compare banding patterns to identify any differences in non-specific binding.

• Application versatility: Test both formats across multiple applications (ELISA, Western blot, immunoprecipitation, immunofluorescence) to determine if one format offers superior performance in specific contexts.

• Multi-label compatibility: Evaluate how each antibody performs in multi-label experiments where multiple proteins are detected simultaneously. Biotinylated antibodies offer advantages for signal amplification but may be limited in multiplexing due to shared detection reagents.

This systematic comparison will help researchers select the optimal antibody format for their specific experimental requirements .

How can MKLN1 Antibody, Biotin conjugated be utilized to study the newly discovered role of MKLN1 in mTORC1 regulation?

Recent discoveries have revealed MKLN1's involvement in mTORC1 regulation, offering exciting new research directions:

• Lysosomal co-localization studies: Utilize the biotinylated MKLN1 antibody with lysosomal markers to investigate whether MKLN1 influences mTORC1 localization to lysosomal membranes, a critical step in mTORC1 activation. Recent research indicates MKLN1 forms a complex with ZMYND19 that blocks a late stage of mTORC1 activation at the lysosomal membrane, independently of the tuberous sclerosis complex .

• Nutrient sensing pathway analysis: Use the biotinylated antibody to monitor MKLN1 localization and protein levels under various nutrient conditions (amino acid starvation/repletion, glucose limitation) to determine if MKLN1 acts as a nutrient-responsive regulator of mTORC1.

• RagA/C and Raptor interaction studies: Implement co-immunoprecipitation with the biotinylated MKLN1 antibody to isolate and characterize complexes containing MKLN1, ZMYND19, Raptor, and RagA/C under different cellular conditions. Research has shown that ZMYND19/MKLN1 bound Raptor and RagA/C .

• Translation rate assessment: Since mTORC1 is a master regulator of protein synthesis, use puromycin incorporation assays in conjunction with MKLN1 detection to correlate MKLN1 levels with translation rates. MAEA knockout (which stabilizes MKLN1) has been shown to diminish translation rate as judged by puromycin chase analysis .

• Rescue experiments: In cells with dysregulated mTORC1 signaling, use the biotinylated antibody to confirm MKLN1 knockdown or overexpression efficiency in experiments designed to rescue normal mTORC1 activity.

These approaches will advance understanding of how CTLH-mediated regulation of MKLN1 stability represents a mechanism for cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway .

What are the potential applications for studying MKLN1 protein turnover dynamics using pulse-chase experiments?

Studying MKLN1 protein turnover dynamics offers valuable insights into its regulation and function:

• Modified SILAC approach: Combine stable isotope labeling with the biotinylated MKLN1 antibody for immunoprecipitation to track newly synthesized versus existing MKLN1 pools, providing precise turnover rates in different cellular contexts.

• Biotin pulse-chase: Leverage the biotinylated antibody in a modified pulse-chase paradigm where cells are pulse-labeled with amino acid analogs (like AHA or HPG) that can be subsequently tagged. This allows discrimination between pre-existing and newly synthesized MKLN1 populations.

• Degradation pathway dissection: Use specific inhibitors of proteasomal (MG132, bortezomib) or lysosomal (bafilomycin A1, chloroquine) degradation in conjunction with the biotinylated antibody to determine the primary pathway responsible for MKLN1 turnover. Research has shown that bortezomib proteasome inhibition increases steady-state MKLN1 levels .

• Half-life determination under varied conditions: Apply cycloheximide chase experiments with detection via the biotinylated antibody to compare MKLN1 half-life under diverse conditions (stress, growth factor stimulation, cell cycle phases). Previous research has established a baseline half-life of approximately 7.7 hours in control cells .

• Co-degradation relationship with ZMYND19: Use double-pulse labeling to simultaneously track MKLN1 and ZMYND19 turnover, as MKLN1 depletion has been shown to increase steady-state ZMYND19 levels, suggesting MKLN1 supports ZMYND19 turnover .

These approaches will provide crucial mechanistic insights into how MKLN1 levels are regulated in various physiological and pathological contexts, potentially revealing new therapeutic targets in conditions where mTOR signaling is dysregulated.

How does MKLN1 Antibody, Biotin conjugated compare with other detection methods for studying MKLN1?

When selecting the optimal approach for MKLN1 detection, it's important to consider the comparative advantages of various methods:

Each method offers distinct advantages depending on the experimental context. The biotinylated antibody excels in applications requiring signal amplification and versatility across multiple detection platforms .

What novel methodological approaches can be combined with MKLN1 Antibody, Biotin conjugated for advanced research applications?

Emerging technologies provide exciting opportunities to enhance MKLN1 research:

• Proximity-dependent biotinylation (BioID/TurboID): Use the biotinylated MKLN1 antibody to validate proximity labeling results from BioID-MKLN1 fusion proteins, providing complementary approaches to mapping MKLN1 protein interaction networks.

• Single-cell Western blotting: Combine microfluidic single-cell capture with on-chip Western blotting using the biotinylated MKLN1 antibody to examine cell-to-cell variation in MKLN1 expression and processing.

• Mass cytometry (CyTOF): Conjugate the MKLN1 antibody with rare earth metals instead of biotin for highly multiplexed protein detection in single cells, allowing simultaneous assessment of dozens of proteins in the MKLN1 regulatory network.

• Super-resolution microscopy: Implement STORM, PALM or expansion microscopy with the biotinylated antibody to achieve nanoscale resolution of MKLN1 localization relative to other cellular structures, particularly in relation to cytoskeletal components and lysosomal membranes where mTORC1 regulation occurs.

• Spatial transcriptomics integration: Combine in situ transcriptomics with protein detection using the biotinylated MKLN1 antibody to correlate MKLN1 protein levels with gene expression patterns at single-cell resolution within tissue contexts.

• Cell-free expression systems: Use the biotinylated antibody to detect and isolate MKLN1 from cell-free expression systems for structural and functional studies, enabling rapid assessment of mutant variants.

These integrated approaches can provide unprecedented insights into MKLN1 biology, particularly its newly discovered roles in mTORC1 regulation and interactions with ZMYND19 .