GLS Antibody

What is GLS and why are antibodies against it important in research?

GLS (Glutaminase) is an enzyme that catalyzes the first reaction in the primary pathway for renal catabolism of glutamine, converting glutamine to glutamate. This conversion supports the tricarboxylic acid cycle and redox and epigenetic reactions . GLS antibodies are critical research tools because:

• They enable detection and quantification of GLS expression in various tissues and cell types

• They help investigate the role of glutaminolysis in cancer metabolism and other diseases

• They support studies into the metabolic reprogramming of cancer cells

• They facilitate research into neurological disorders, as GLS regulates levels of glutamate, the main excitatory neurotransmitter in the brain

What are the different isoforms of GLS that antibodies might target?

GLS has multiple isoforms produced by alternative splicing that researchers should consider when selecting antibodies:

Some antibodies like Proteintech's 23549-1-AP can recognize all three isoforms (KGA, GAM, GAC) of GLS , which is an important consideration when designing experiments to study specific or multiple isoforms.

How should researchers optimize GLS antibody performance in Western blotting?

Optimizing GLS antibody performance in Western blotting requires attention to several technical details:

• Expected molecular weight considerations: GLS has a calculated molecular weight of 73 kDa, but observed bands may appear at 58-65 kDa depending on the isoform . This discrepancy is normal and has been validated in published studies.

• Sample preparation protocol:

  • Use fresh tissue/cells and maintain cold conditions throughout

  • Include protease inhibitors in lysis buffer to prevent degradation

  • Consider phosphatase inhibitors as GLS activity can be regulated by phosphorylation

  • For mitochondrial proteins like GLS, ensure proper subcellular fractionation if studying specific compartments

• Antibody validation strategy:

  • Verify specificity using positive control tissues (brain, kidney, liver)

  • Include appropriate negative controls

  • Consider validation through knockdown/knockout samples when available

• Recommended blocking conditions: 5% non-fat milk in TBST is typically effective, though specific antibodies may have optimized conditions in their datasheets .

How can researchers ensure reproducible results when using GLS antibodies for immunohistochemistry?

To ensure reproducibility in immunohistochemistry with GLS antibodies:

• Antigen retrieval optimization: For GLS antibodies, suggested antigen retrieval includes:

  • TE buffer pH 9.0 often provides optimal results

  • Citrate buffer pH 6.0 is an alternative method

  • Test both conditions to determine which works best for your specific tissue

• Fixation considerations:

  • PFA fixation has been validated for GLS detection in multiple cell lines

  • Overfixation can mask epitopes; follow validated protocols for fixation times

  • For frozen sections, cold acetone or methanol fixation may preserve antigenicity

• Signal amplification methods:

  • For low-expressing samples, consider tyramide signal amplification

  • Biotin-based amplification systems should be used cautiously as endogenous biotin can create background in metabolically active tissues

• Validation across multiple tissues:

  • GLS antibodies have been validated in human kidney and brain tissues

  • Always include known positive controls in each experimental run

What are the critical factors for selecting the optimal GLS antibody for a specific research application?

When selecting a GLS antibody for specific research applications, consider:

• Epitope location relative to functional domains:

  • Some antibodies target the C-terminus region (e.g., ab260047 targets aa 600 to C-terminus)

  • Others target fusion proteins containing specific sequences (e.g., GLS fusion protein Ag31167)

  • For studying specific functions, choose antibodies that don't interfere with functional domains

• Cross-reactivity with species of interest:

  Antibody Catalog #	Tested Reactivity	Cited Reactivity	Reference
  29519-1-AP	Human	Human, mouse, rat
  CAB3885	Human, mouse, rat	-
  ab260047	Human	-

• Validation data availability:

  • Review validation data galleries provided by manufacturers

  • Examine published literature citing specific antibody clones

  • Assess reproducibility across multiple cell lines and tissues

• Ability to distinguish between isoforms:

  • Some antibodies recognize all GLS isoforms

  • Others may be isoform-specific

  • Select based on whether your research questions require isoform discrimination

How can GLS antibodies be used to investigate metabolic reprogramming in cancer cells?

GLS antibodies are instrumental in studying cancer metabolism:

• Comparative expression analysis techniques:

  • Use immunoblotting to quantify GLS expression across cancer cell lines

  • The glutaminase band in AA/C1 cells is more intense than in HT29 cells, correlating with glutaminase activity measurements

  • GLS expression has been shown to increase ~10-fold in response to Myc in certain cancer contexts

• Co-localization studies:

  • Immunofluorescence using GLS antibodies can reveal subcellular localization changes in cancer cells

  • Combined with mitochondrial markers, researchers can study altered compartmentalization

• Patient sample stratification:

  • GLS expression correlates with prognosis in multiple cancer types

  • High GLS expression is associated with poor survival in COAD, HNSC, KIRP, LAML, LGG, LIHC, STAD, and UCEC

• Experimental models:

  • GLS antibodies have been validated in multiple cancer cell lines including COLO 320, A549, HeLa, MCF-7, and SH-SY5Y

  • These models can be used to study the effects of glutaminase inhibitors

What is the relationship between GLS expression and tumor immune microenvironment, and how can antibodies help investigate this?

GLS antibodies can help investigate the complex relationship between GLS expression and the tumor immune microenvironment:

• Correlation with immune cell infiltration:
  High GLS expression correlates with altered immune cell infiltration patterns:

  • Decreased infiltration of B cells, T cells, CD8+ T cells, Th17 cells, DCs, NK cells, and cytotoxic cells

  • Increased infiltration of Th2 cells, which typically promote tumor growth

• Multi-parameter immunofluorescence techniques:

  • Combined staining with GLS antibodies and immune cell markers can reveal spatial relationships

  • This approach can map metabolic zones within tumors and their impact on immune cell function

• Mechanistic studies of metabolic immune suppression:

  • GLS expression negatively correlates with antineoplastic Th1 cell infiltration across almost all tumors

  • It positively correlates with cancer-promoting Th2 cell infiltration and with cancer-associated fibroblast (CAF) infiltration

• Immunotherapy response prediction:

  • High GLS expression is associated with lower MSI, TMB, and neoantigen count in multiple cancer types

  • This correlates with potentially weaker response to immune checkpoint inhibitor therapy

  • In COAD, STAD, UCEC, SKCM, and OV, high GLS expression predicts poorer response to various immunotherapeutic approaches

How can researchers use GLS antibodies to evaluate the efficacy of glutaminase inhibitors in cancer therapy?

GLS antibodies are essential tools for evaluating glutaminase inhibitor efficacy:

• Target engagement assessment:

  • Western blotting with GLS antibodies can confirm that inhibitors are reaching their target

  • Changes in post-translational modifications can be monitored using phospho-specific antibodies

• Pharmacodynamic marker development:

  • IHC staining of tumor biopsies pre- and post-treatment can measure GLS levels as a pharmacodynamic marker

  • Combined with downstream metabolite measurements, this creates a comprehensive view of drug activity

• Resistance mechanism investigation:

  • In resistant models, GLS antibodies can help identify changes in expression, localization, or isoform switching

  • Immunoprecipitation followed by mass spectrometry can identify novel binding partners in resistant contexts

• Combination therapy rationale development:

  • IHC co-staining of GLS with other metabolic enzymes can identify patients who might benefit from combination approaches

  • For example, tumors with both high GLS and high PD-L1 might benefit from combined glutaminase inhibitors and immune checkpoint inhibitors

How does GLS regulate T cell differentiation and function, and what methods should researchers use to study this?

Research has revealed critical roles for GLS in T cell biology that can be studied using GLS antibodies:

• GLS knockout and inhibition studies:

  • GLS-deficient T cells show increased IFNγ expression in Th1-skewing conditions

  • Conversely, they show decreased IL17A expression in Th17-skewing conditions

  • This differential effect is linked to transcription factor expression patterns, with increased T-bet in Th1 and decreased RORγt in Th17 conditions

• Metabolic flux analysis:

  • By day 5 of differentiation, Th1 cells with GLS inhibition show increased glucose uptake and glycolytic flux

  • In contrast, Th17 cells remain metabolically impaired by GLS inhibition

  • These changes can be correlated with antibody-based measurements of GLS protein levels

• Temporal analysis of metabolic adaptation:

  • During early activation (days 1-2), both Th1 and Th17 cells show reduced transcription factors and cell size with GLS inhibition

  • By day 5, Th1 cells recover and show increased cell size and T-bet expression

  • Total rRNA levels reflect these changes, with similar levels at day 3 but divergent patterns by day 5

• Methodology considerations:

  • Use flow cytometry with GLS antibodies to quantify GLS levels at single-cell resolution during differentiation

  • Combine with metabolic dyes to correlate GLS expression with functional metabolic states

  • Consider subcellular fractionation to track potential redistribution during activation

What are the technical challenges in using GLS antibodies for flow cytometry in immune cell analysis?

Flow cytometry with GLS antibodies presents several technical challenges:

• Intracellular staining optimization:

  • GLS is primarily located in mitochondria, requiring effective permeabilization

  • Standard paraformaldehyde fixation followed by saponin or Triton X-100 permeabilization works for most applications

  • For co-staining with surface markers, use a sequential staining approach: surface markers first, followed by fixation, permeabilization, and GLS antibody staining

• Signal-to-noise considerations:

  • Autofluorescence can be a challenge in metabolically active cells with high mitochondrial content

  • Use appropriate fluorochromes that excite/emit away from cellular autofluorescence peaks

  • Include FMO (fluorescence minus one) controls to set accurate gates

• Epitope preservation:

  • Some fixation methods may mask GLS epitopes

  • Test multiple permeabilization reagents (e.g., Triton X-100, saponin, methanol) to determine optimal conditions

  • Consider using indirect staining with secondary antibodies for signal amplification

• Validation strategies:

  • Use GLS knockout or knockdown cells as negative controls

  • Compare expression patterns with known GLS expression data across immune cell types

  • Confirm flow cytometry results with other methods (e.g., Western blot, qPCR)

How does GLS expression in the tumor microenvironment affect immunotherapy response, and what research methods can investigate this relationship?

The relationship between GLS expression and immunotherapy response can be investigated through several approaches:

• Multiplex immunohistochemistry:

  • Combine GLS antibodies with immune checkpoint markers (PD-1, PD-L1, CTLA-4)

  • Quantify spatial relationships between GLS-high tumor regions and immune infiltrates

  • This approach reveals the metabolic landscape that may affect immunotherapy efficacy

• Correlation with immunotherapy response biomarkers:

  • High GLS expression correlates with:

    • Lower microsatellite instability (MSI) in COAD, HNSC, DLBC, KICH, and PRAD

    • Lower tumor mutation burden (TMB) in COAD, STAD, and CHOL

    • Fewer neoantigens in COAD and CHOL

  • These factors are known predictors of immune checkpoint inhibitor response

• Immunophenotyping studies:

  • GLS expression correlates with specific changes in tumor immune microenvironment:

  • Negative correlation with CD8+ T lymphocyte abundance in HNSC, UCEC, and CESC

  • Positive correlation with cancer-associated fibroblast infiltration across almost all tumors

  • Positive correlation with immunosuppressive MDSC infiltration in COAD, HNSC, LGG, LIHC, and UCEC

• Combination therapy research models:

  • For tumors with high GLS expression, combining glutaminase inhibitors with immune checkpoint inhibitors may overcome resistance

  • Mouse models with humanized immune systems can be used to test such combinations

  • GLS antibodies are essential tools to monitor on-target effects in these models

How can researchers address inconsistent molecular weight observations when detecting GLS by Western blot?

Inconsistent molecular weight observations with GLS antibodies can be addressed methodically:

• Understanding expected weight variations:

  • The calculated molecular weight of GLS is 73 kDa

  • Observed molecular weights often range from 58-65 kDa

  • Different isoforms (KGA, GAC, GAM) may appear at different molecular weights

• Post-translational modification considerations:

  • Phosphorylation of GLS can alter its mobility on SDS-PAGE

  • Proteolytic processing may generate fragments

  • Try phosphatase treatment of samples to determine if modifications affect migration

• Sample preparation adjustments:

  • Use freshly prepared samples when possible

  • Include multiple protease inhibitors in lysis buffer

  • Avoid freeze-thaw cycles that may lead to degradation

  • Consider non-reducing conditions if disulfide bonds affect epitope recognition

• Gel system optimization:

  • Use gradient gels (4-20%) to better resolve proteins across a wide molecular weight range

  • Adjust running conditions (voltage, time) to improve resolution in the relevant size range

  • Consider native PAGE if protein folding affects antibody recognition

What are the most common causes of non-specific staining with GLS antibodies and how can they be mitigated?

Non-specific staining with GLS antibodies can be addressed through several approaches:

• Blocking optimization:

  • Test different blocking solutions (BSA, normal serum, commercial blockers)

  • Extend blocking time to reduce background

  • Use the blocking solution as antibody diluent to maintain blocking during incubation

• Antibody validation and specificity:

  • Verify antibody specificity through knockout/knockdown controls

  • Peptide competition assays can confirm specificity

  • Pre-adsorption of the antibody with recombinant antigen can reduce non-specific binding

• Sample-specific challenges:

  • Some tissues (e.g., liver, kidney) may have high endogenous biotin, causing background with biotin-based detection systems

  • Endogenous peroxidase activity in tissues like spleen can cause background with HRP-based systems

  • Use appropriate quenching steps (hydrogen peroxide treatment for peroxidase, avidin/biotin blocking for biotin)

• Protocol modifications:

  • Increase washing steps in number and duration

  • Add detergent (0.1-0.3% Triton X-100) to reduce hydrophobic interactions

  • Titrate primary antibody to find optimal concentration balancing signal and background

  • For immunofluorescence, include an autofluorescence quenching step

How should researchers interpret and validate contradictory findings when using different GLS antibodies?

When faced with contradictory results from different GLS antibodies:

• Epitope mapping and comparison:

  • Identify the epitopes recognized by each antibody:

    • CAB3885 targets amino acids 610-669 of human GLS

    • ab260047 targets amino acids 600 to C-terminus

    • 29519-1-AP is raised against GLS fusion protein Ag31167

  • Different epitopes may be differentially accessible in various experimental conditions

• Validation through orthogonal methods:

  • Confirm protein expression using mRNA analysis (qPCR, RNA-seq)

  • Use mass spectrometry-based proteomics for unbiased protein detection

  • Apply genetic approaches (siRNA, CRISPR) to validate antibody specificity

• Isoform-specific considerations:

  • Determine if contradictory results stem from isoform-specific detection

  • Use isoform-specific primers in qPCR to correlate with protein findings

  • Consider that different isoforms may predominate in different tissues or conditions

• Documentation and reporting practices:

  • Thoroughly document all experimental conditions

  • Report the specific antibody clone, catalog number, and lot in publications

  • Include detailed methods sections describing validation approaches

  • Contact antibody manufacturers with contradictory findings to contribute to knowledge base

How can researchers use GLS antibodies in combination with single-cell technologies to study metabolic heterogeneity?

Combining GLS antibodies with single-cell technologies enables powerful analyses of metabolic heterogeneity:

• Single-cell Western blotting approaches:

  • Microfluidic platforms can perform Western blots on individual cells

  • GLS antibodies can quantify expression at single-cell resolution

  • This reveals population heterogeneity masked in bulk analyses

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated GLS antibodies can be incorporated into CyTOF panels

  • Combined with functional markers, this approach can correlate GLS expression with cell state

  • Allows simultaneous measurement of up to 40 parameters, including multiple metabolic enzymes

• Spatial transcriptomics correlation:

  • GLS antibody staining on serial sections can be correlated with spatial transcriptomics data

  • This approach maps metabolic zones within tumors or tissues

  • Reveals relationships between GLS expression and local microenvironmental features

• Methodological considerations:

  • Validate antibodies specifically for single-cell applications

  • Include appropriate single-cell controls

  • Consider fixation and permeabilization conditions that preserve both target epitopes and cellular morphology

What are the considerations for using GLS antibodies in developing companion diagnostics for glutaminase inhibitor therapies?

Developing companion diagnostics with GLS antibodies requires specific considerations:

• Standardization requirements:

  • Rigorous antibody validation across multiple laboratories

  • Establishment of scoring systems for quantitative assessment

  • Development of reference standards for calibration

• Predictive biomarker selection:

  • Determine whether total GLS protein levels, specific isoforms, or post-translational modifications best predict response

  • Correlate IHC findings with functional metabolic measurements

  • Develop multi-parameter predictive algorithms that may include GLS along with other markers

• Technical standardization for clinical implementation:

  • Automated staining platforms to ensure reproducibility

  • Digital pathology approaches for objective quantification

  • Standard operating procedures that can be implemented across clinical laboratories

• Validation in clinical trial contexts:

  • Retrospective analysis of samples from glutaminase inhibitor trials

  • Prospective collection in basket trials to correlate expression with response

  • Establishment of clinically meaningful cutoffs for "high" versus "low" expression

How can researchers investigate the relationship between GLS and other metabolic enzymes using antibody-based approaches?

Investigating metabolic enzyme networks using GLS antibodies involves several approaches:

• Co-immunoprecipitation studies:

  • Use GLS antibodies for immunoprecipitation followed by mass spectrometry

  • Identify novel interaction partners in different cellular contexts

  • Validate findings with reciprocal co-IPs using antibodies against identified partners

• Multiplex immunofluorescence techniques:

  • Combine GLS antibodies with antibodies against other metabolic enzymes

  • This approach reveals spatial relationships and potential metabolic compartmentalization

  • Quantify co-localization using digital image analysis

• Proximity ligation assay (PLA):

  • Use PLA to detect proteins in close proximity (<40 nm)

  • This technique can reveal transient or weak interactions between GLS and other proteins

  • Provides spatial resolution beyond conventional co-localization studies

• Chromatin immunoprecipitation (ChIP) studies:

  • Investigate whether GLS or its metabolic products affect transcriptional regulation

  • Use antibodies against histone modifications affected by glutamine metabolism

  • Correlate findings with GLS expression and activity levels