THEMIS Recombinant Monoclonal Antibody

What is THEMIS and what is its role in T-cell development?

THEMIS (thymocyte-expressed molecule involved in selection), also called Gasp (Grb2-associating protein), is a 72-73 kDa cytoplasmic phosphoprotein that plays a central role in late thymocyte development. It controls both positive and negative T-cell selection processes in the thymus and is required to sustain and integrate signals necessary for proper lineage commitment and maturation of T-cells .

The protein functions primarily by regulating T-cell development through T-cell antigen receptor (TCR) signaling, particularly through the regulation of calcium influx and phosphorylation of Erk . THEMIS has been found to associate with several signaling molecules including Grb2, PLC gamma 1, and LAT, and is quickly phosphorylated by SLP-76 following TCR signaling . Highest expression levels are found in CD4+CD8+ double-positive thymocytes, with lower expression in mature T cells .

What applications are THEMIS recombinant monoclonal antibodies validated for?

THEMIS recombinant monoclonal antibodies have been validated for multiple research applications, including:

Many of these antibodies have demonstrated effectiveness in detecting THEMIS across multiple species, with some showing cross-reactivity between human, mouse, and rat samples .

How should I select the appropriate THEMIS antibody for my specific research needs?

Selecting the appropriate THEMIS antibody requires consideration of several key factors:

Species reactivity and cross-reactivity

Determine whether you need an antibody that recognizes human, mouse, or rat THEMIS. Some antibodies show cross-reactivity across species, while others are species-specific. For example, clone 719945 shows 100% cross-reactivity with recombinant human THEMIS under non-reducing conditions but only approximately 10% cross-reactivity under reducing conditions .

Application compatibility

Ensure the antibody is validated for your intended application. Some THEMIS antibodies perform well across multiple applications, while others are optimized for specific techniques:

• Clone EPR7353 (ab126771) is suitable for ICC/IF, IP, WB, and Flow Cytometry

• Clone 7G7 is recommended for ELISA, IHC, IF, and FC applications

• Clone 719945 is validated for Flow Cytometry and Western Blot

Epitope recognition

Different antibodies target different regions of the THEMIS protein:

• Clone 719945 targets mouse THEMIS region Lys122-Lys237

• Clone 7G7 was raised against a synthesized peptide derived from Human THEMIS

Validation data

Review the available experimental data showing the antibody's performance in applications similar to yours. Look for evidence of specificity, such as Western blot images showing a single band at the expected molecular weight (approximately 72-73 kDa for THEMIS) .

What are the optimal storage conditions for THEMIS recombinant monoclonal antibodies?

Based on manufacturer recommendations from multiple sources, the following storage conditions are optimal for maintaining THEMIS antibody stability and function:

Temperature conditions

• Long-term storage: -20°C to -80°C

• For working solutions: 2-8°C for up to 1 month under sterile conditions

• Some antibodies specifically recommend -80°C for preferred long-term storage

Buffer composition and formulation

Most THEMIS antibodies are supplied in one of the following formulations:

• Liquid form in PBS with 50% glycerol and 0.02% sodium azide, pH 7.4

• Lyophilized from PBS solution, often with trehalose as a cryoprotectant

Handling recommendations

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• For lyophilized products, reconstitute in sterile PBS to a final concentration of 0.5 mg/mL

• Allow complete dissolution before use

• Aliquot immediately after reconstitution to minimize freeze-thaw damage

Stability timeframes

Based on manufacturer data, typical stability periods include:

• 12 months from date of receipt at -20°C to -70°C as supplied

• 1 month at 2-8°C under sterile conditions after reconstitution

• 6 months at -20°C to -70°C under sterile conditions after reconstitution

How can I optimize Western blot protocols for detecting THEMIS?

Optimizing Western blot protocols for THEMIS detection requires attention to several critical parameters:

Sample preparation

• Use tissue samples with known THEMIS expression (e.g., thymus, Jurkat cells)

• Extract proteins using RIPA or NP-40 based lysis buffers containing protease inhibitors

• Maintain samples on ice during processing to minimize protein degradation

• Use appropriate protein quantification methods to ensure equal loading

Gel electrophoresis conditions

• Use 8-10% polyacrylamide gels to properly resolve the 72-73 kDa THEMIS protein

• Load 10-20 μg of total protein per lane

• Include positive controls (e.g., mouse thymus tissue, Jurkat cell lysate)

• Run in reducing conditions, but note that some antibodies may perform differently under non-reducing conditions

Transfer and detection

• Use PVDF membranes for optimal protein binding

• Block with 5% non-fat dry milk in TBST (or according to antibody manufacturer's recommendations)

• Optimal primary antibody dilutions vary by clone:

  • EPR7354: 1/10000 dilution

  • EPR7353: 1/1000 dilution

  • 719945: 1 μg/mL

• Use appropriate HRP-conjugated secondary antibodies and detection systems

Validated protocol example

From experimental data in search result :

• Sample: Jurkat or MOLT-4 cell lysate (20 μg)

• Antibody: Anti-Themis antibody [EPR7353] at 1/1000 dilution

• Secondary: Goat Anti-Rabbit IgG H&L (HRP) at 1/20000 dilution

• Expected result: Single band at approximately 73 kDa

What are the best practices for intracellular THEMIS staining in flow cytometry?

For successful intracellular THEMIS staining in flow cytometry, follow these research-validated procedures:

Cell preparation and fixation

• Isolate cells of interest (e.g., thymocytes, peripheral blood lymphocytes, or T cell lines)

• Wash cells thoroughly in PBS containing 2% FBS or similar buffer

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Some protocols specifically mention using paraformaldehyde fixation for THEMIS detection

Permeabilization methods

Several effective permeabilization approaches have been documented:

• Saponin (0.1-0.5%): Enables access to cytoplasmic antigens while preserving membrane structure

• Triton X-100 (0.1-0.2%): More aggressive permeabilization for some applications

• Methanol (90%): Alternative approach mentioned in some protocols

Staining protocol

• Block with 10% normal serum (from the same species as secondary antibody) to reduce background

• Incubate with primary THEMIS antibody:

  • Clone 719945 has been used successfully for intracellular flow cytometry

  • Clone EPR7353 has been used at 1/50 dilution (10 μg/ml)

• Wash thoroughly and incubate with appropriate fluorochrome-conjugated secondary antibody

• Include proper isotype controls to determine specific staining

Validated protocol example

From search result :
"Mouse thymocytes were stained with Rat Anti-Mouse Themis Monoclonal Antibody (filled histogram) or isotype control antibody (open histogram), followed by Phycoerythrin-conjugated Anti-Rat IgG Secondary Antibody. To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin."

How can I troubleshoot cross-reactivity and specificity issues with THEMIS antibodies?

Addressing cross-reactivity and specificity challenges with THEMIS antibodies requires systematic troubleshooting approaches:

Verifying antibody specificity

• Test antibodies in THEMIS knockout or knockdown models to confirm specificity

• Compare staining patterns across tissues (THEMIS expression should be primarily in thymus and T cells)

• Use multiple antibodies targeting different THEMIS epitopes to corroborate findings

Optimizing experimental conditions

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Test different blocking reagents (BSA, normal serum, commercial blockers) to reduce non-specific binding

• Adjust washing stringency (buffer composition, number of washes, detergent concentration)

• Consider native versus denatured conditions, as this impacts epitope accessibility

Species cross-reactivity considerations

Some THEMIS antibodies show different cross-reactivity patterns under various conditions:

• Clone 719945: "100% cross-reactivity with recombinant human THEMIS (aa 2-282) under non-reducing conditions, and approximately 10% cross-reactivity under reducing conditions"

• Other antibodies like EPR7353 have been validated to work effectively across human, mouse, and rat samples

Application-specific modifications

• Western blot: Optimize reducing vs. non-reducing conditions

• Flow cytometry: Test different fixation and permeabilization methods

• IHC/ICC: Evaluate various antigen retrieval techniques (heat-induced vs. enzymatic)

Controls to include

• Positive controls: Tissues or cells known to express THEMIS (thymus, Jurkat cells)

• Negative controls: Non-T cell lineages or THEMIS-negative tissues

• Technical controls: Secondary antibody only, isotype controls, blocking peptide competition

What methods are effective for characterizing post-translational modifications of THEMIS?

Characterizing post-translational modifications (PTMs) of THEMIS requires sophisticated analytical approaches:

Mass spectrometry-based strategies

Recent advances in multidimensional LC/MS approaches provide powerful tools for characterizing THEMIS PTMs:

• Bottom-up proteomics: Digest THEMIS into peptides and analyze by LC-MS/MS

• Middle-down proteomics: Analyze larger THEMIS fragments

• Top-down proteomics: Analyze intact THEMIS protein

A particularly effective approach based on search result includes:
"A four-dimensional-liquid chromatography/mass spectrometry (4D-LC/MS) method...for on-line digestion, followed by...reduction and fragments separation...This strategy considerably improved the on-line digestion efficiency with higher sequence coverages (LC and HC >97%), thus allowing various PTMs including oxidation, deamidation, and isomerization...to be monitored"

Phosphorylation analysis

Given THEMIS's role in TCR signaling, phosphorylation analysis is particularly relevant:

• Immunoprecipitate THEMIS followed by phospho-specific Western blotting

• Use phospho-enrichment techniques (IMAC, TiO2) prior to MS analysis

• Apply Phos-tag SDS-PAGE to separate phosphorylated THEMIS forms

• Consider comparing baseline vs. TCR-stimulated conditions to capture signaling-dependent phosphorylation events

Sample preparation considerations

• Preserve PTMs during extraction by including appropriate inhibitors (phosphatase inhibitors, deubiquitinase inhibitors)

• Consider cell stimulation conditions that promote specific modifications (e.g., TCR activation)

• Compare THEMIS PTM profiles across different developmental stages of T cells

Validation approaches

• Functional studies using PTM-deficient THEMIS mutants

• Temporal analysis of modifications in response to signaling events

• Correlation of PTM patterns with phenotypic outcomes in T cell development models

How can I design co-immunoprecipitation experiments to identify THEMIS binding partners?

Designing effective co-immunoprecipitation (co-IP) experiments for THEMIS interaction studies requires careful consideration of experimental parameters:

Experimental design

• Cell selection: Use cells with documented THEMIS expression (Jurkat cells, thymocytes)

• Stimulation conditions: Compare resting vs. TCR-stimulated states to capture dynamics of protein interactions

• Controls: Include isotype control antibodies and THEMIS-deficient cells as negative controls

Lysis buffer optimization

• Use gentle non-ionic detergents (NP-40, Triton X-100) to preserve protein-protein interactions

• Include protease and phosphatase inhibitors to maintain interaction integrity

• Test different salt concentrations to balance specificity with maintenance of interactions

Immunoprecipitation procedure

Based on validated protocols from search result :

• Prepare cell lysate (e.g., Jurkat whole cell lysate)

• Use a validated THEMIS antibody with demonstrated IP capability (e.g., ab126771 at 1/30 dilution, 2μg)

• Include negative control (e.g., Rabbit monoclonal IgG isotype control)

• Use appropriate detection reagents (e.g., VeriBlot for IP Detection Reagent)

Detection of binding partners

• Western blot analysis: Probe for known or suspected interaction partners (Grb2, PLC gamma 1, LAT)

• Mass spectrometry: For unbiased identification of the complete THEMIS interactome

• Reverse co-IP validation: Immunoprecipitate identified binding partners and probe for THEMIS

Example protocol parameters

From search result :
"Purified ab126771 at 1/30 dilution (2μg) immunoprecipitating Themis in Jurkat whole cell lysate.
Lane 1 (input): Jurkat whole cell lysate 10μg
Lane 2 (+): ab126771 + Jurkat whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG instead of ab126771 in Jurkat whole cell lysate
VeriBlot for IP Detection Reagent (HRP) (1/1000 dilution) was used for Western blotting"

What approaches are available for validating THEMIS antibody specificity?

Comprehensive validation of THEMIS antibody specificity requires multiple complementary approaches:

Genetic validation methods

• THEMIS knockout models: The gold standard for specificity validation

• THEMIS knockdown using siRNA or shRNA: Alternative when knockout models aren't available

• Overexpression systems: Using tagged THEMIS constructs as positive controls

Biochemical validation approaches

• Western blot analysis: Should show a single band at the expected molecular weight (72-73 kDa)

• Peptide competition assays: Pre-incubation with immunizing peptide should block specific binding

• Multiple antibody comparison: Different antibodies against distinct THEMIS epitopes should show similar patterns

Advanced validation strategies

• Immunoprecipitation followed by mass spectrometry: Confirms pull-down of THEMIS protein

• Immunodepletion: Sequential immunoprecipitation should progressively deplete THEMIS signal

• Surface plasmon resonance or ELISA: Quantitative assessment of binding kinetics and specificity

Validation across applications

Comprehensive validation should include multiple techniques:

• Western blot: For expression level analysis and molecular weight confirmation

• Flow cytometry: For expression patterns in single cells

• Immunohistochemistry: For tissue localization patterns

• Immunoprecipitation: For functional protein interaction studies

Example validation data

From search result : "Western blot shows lysates of mouse thymus tissue. PVDF membrane was probed with 1 µg/mL of Rat Anti-Mouse Themis Monoclonal Antibody followed by HRP-conjugated Anti-Rat IgG Secondary Antibody. A specific band was detected for Themis at approximately 72 kDa. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1."