PGM2L1 Antibody

What is PGM2L1 and what is its primary function in cellular metabolism?

PGM2L1 (phosphoglucomutase 2-like 1) functions as a glucose 1,6-bisphosphate synthase that uses 1,3-bisphosphoglycerate as a phosphate donor and a series of 1-phosphate sugars as acceptors, including glucose 1-phosphate, mannose 1-phosphate, ribose 1-phosphate, and deoxyribose 1-phosphate . Interestingly, while 5 or 6-phosphosugars are generally poor substrates, glucose 6-phosphate is an exception . PGM2L1 also synthesizes ribose 1,5-bisphosphate and exhibits low phosphopentomutase and phosphoglucomutase activities . Research has shown that PGM2L1 is tailored to raise the concentration of Glc-1,6-P2 to high values, unlike PGM2 which has intrinsically lower Glc-1,6-P2 synthase activity and is more strongly inhibited by the reaction product Glc-1,6-P2 .

How should I select the most appropriate PGM2L1 antibody for my specific experimental requirements?

When selecting a PGM2L1 antibody, consider the following methodology:

• Define your application requirements: Different antibodies are validated for specific applications. For example, if conducting immunohistochemistry and western blot, Boster Bio's OTI5F5 clone (M12656-1) has been validated for both applications , while some antibodies like OTI2D12 (M12656) are validated only for western blot .

• Species reactivity considerations: Verify that the antibody reacts with your target species. Most commercial PGM2L1 antibodies react with human, mouse, and rat samples .

• Clonality selection:

  • Monoclonal antibodies (e.g., OTI5F5, OTI4G6) provide high specificity and reproducibility between experiments

  • Polyclonal antibodies (e.g., Proteintech 13942-1-AP) may offer higher sensitivity by recognizing multiple epitopes

• Validation data assessment: Review the manufacturer's validation images and protocols. For instance, Boster validates all antibodies on WB, IHC, ICC, Immunofluorescence, and ELISA with known positive control and negative samples .

• Molecular weight confirmation: Ensure the antibody detects the expected molecular weight of PGM2L1 (approximately 70.3 kDa) .

What dilution ranges are recommended for different applications of PGM2L1 antibodies?

Based on manufacturer recommendations and validation data, the following dilution ranges are suggested:

Note that these are starting points for optimization, and the actual working concentration may vary based on your specific experimental conditions and sample types .

What validation methods should I use to confirm PGM2L1 antibody specificity?

To rigorously validate PGM2L1 antibody specificity, implement the following methodological approach:

• Positive and negative controls:

  • Use HEK293T cells transfected with pCMV6-ENTRY PGM2L1 as a positive control alongside empty vector-transfected cells as a negative control, as demonstrated in validation images from multiple manufacturers .

  • Include tissue samples known to express PGM2L1 (brain tissue, skeletal muscle) as positive controls .

• Knockdown/knockout validation:

  • Implement siRNA knockdown or CRISPR/Cas9 knockout of PGM2L1 to demonstrate specificity via signal reduction or elimination.

  • Review published KD/KO validation data where available (e.g., Proteintech reports KD/KO validation in published applications) .

• Molecular weight verification:

  • Confirm the detected protein band appears at approximately 70-72 kDa, matching the calculated molecular weight of PGM2L1 (70.279 kDa) .

• Cross-reactivity assessment:

  • Test the antibody against related proteins (e.g., other PGM family members) to ensure specificity.

  • Consider using tissue panels where PGM2L1 expression is well-characterized.

• Multiple antibody approach:

  • Validate results using antibodies from different manufacturers or those targeting different epitopes of the same protein.

How can I interpret the significance of discrepancies in molecular weight detection between predicted and observed values for PGM2L1?

When addressing molecular weight discrepancies between predicted and observed values for PGM2L1, consider the following analytical framework:

• Expected versus observed weights:

  • The calculated molecular weight of PGM2L1 is approximately 70.279 kDa .

  • Proteintech reports an observed molecular weight of 72 kDa for their antibody .

  • This slight discrepancy (~2 kDa) falls within acceptable ranges for SDS-PAGE variation.

• Factors affecting migration patterns:

  • Post-translational modifications: Phosphorylation (particularly relevant as PGM2L1 functions in phosphate transfer reactions) can add approximately 80 Da per phosphorylation site.

  • Protein folding and structural elements: Some protein domains may resist complete denaturation in SDS-PAGE.

  • Charge distribution: Unusual amino acid compositions can affect migration.

• Experimental verification approach:

  • Run recombinant PGM2L1 protein (e.g., ABIN3094489 which contains AA 1-622 with Strep Tag) alongside your samples.

  • Test with multiple antibodies recognizing different epitopes of PGM2L1.

  • Consider treating samples with phosphatase to determine if post-translational modifications contribute to migration differences.

• Functional context:

  • Research has shown that PGM2L1 can appear as a band-shift in response to phosphorylation events, particularly by AMPK , which may account for observed weight differences in various experimental contexts.

What are the optimal conditions for Western blot analysis using PGM2L1 antibodies?

For optimal Western blot analysis with PGM2L1 antibodies, implement the following protocol guidelines:

• Sample preparation:

  • For cell lysates: Use RIPA buffer with protease and phosphatase inhibitors.

  • For tissue samples: Mouse brain tissue, skeletal muscle, cerebellum, and rat brain tissue have shown positive Western blot detection with PGM2L1 antibodies .

  • Load 20-30 μg of total protein per lane.

• Gel electrophoresis parameters:

  • Use 10% SDS-PAGE gels to achieve optimal separation around the 70 kDa range.

  • Include molecular weight markers spanning 50-100 kDa range.

• Transfer conditions:

  • Use PVDF membranes for optimal protein retention.

  • Transfer at 100V for 60-90 minutes or 30V overnight at 4°C.

• Blocking parameters:

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

• Antibody incubation:

  • Primary antibody dilutions:

    • OTI5F5 (M12656-1): 1:2000

    • OTI2D12 (M12656): 1:500-1:2000

    • Proteintech 13942-1-AP: 1:1000-1:6000

  • Incubate primary antibody overnight at 4°C.

  • Secondary antibody: HRP-conjugated anti-species antibody at 1:5000-1:10000 for 1 hour at room temperature.

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) detection systems.

  • For low expression samples, consider using high-sensitivity ECL reagents.

• Positive control recommendation:

  • HEK293T cells transfected with pCMV6-ENTRY PGM2L1 have been demonstrated as effective positive controls in validation studies .

How can I optimize immunohistochemistry protocols for PGM2L1 detection in tissue samples?

To optimize immunohistochemistry protocols for PGM2L1 detection in tissue samples, follow these detailed methodological guidelines:

• Tissue preparation and fixation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours.

  • Process and embed in paraffin following standard histological procedures.

  • Section tissues at 4-5 μm thickness.

• Antigen retrieval optimization:

  • For Proteintech 13942-1-AP antibody, use TE buffer pH 9.0 for antigen retrieval .

  • Alternative approach: Use citrate buffer pH 6.0 for antigen retrieval .

  • Heat-induced epitope retrieval: Pressure cooker or microwave heating for 10-20 minutes.

• Blocking parameters:

  • Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes.

  • Block non-specific binding with 5-10% normal serum from the same species as the secondary antibody.

• Antibody dilution and incubation:

  • For OTI5F5 (M12656-1): Use at 1:500 dilution .

  • For Proteintech 13942-1-AP: Use at 1:50-1:500 dilution .

  • Incubate primary antibody overnight at 4°C in a humidified chamber.

  • Incubate appropriate HRP-conjugated secondary antibody for 30-60 minutes at room temperature.

• Detection system selection:

  • Use DAB (3,3'-diaminobenzidine) as chromogen for visualization.

  • Counterstain with hematoxylin for nuclear detail.

• Validated positive control tissues:

  • Human gliomas tissue has been validated for positive IHC detection of PGM2L1 .

  • Consider including brain tissue sections as positive controls based on known expression patterns.

• Negative controls:

  • Omit primary antibody or use isotype-matched control antibodies.

  • Consider tissues known to have low PGM2L1 expression based on tissue expression databases.

Troubleshooting and Advanced Applications

To effectively investigate the interplay between PGM2L1 and PMM1 in glucose metabolism, implement this research methodology:

• Co-immunoprecipitation approaches:

  • Use Proteintech 13942-1-AP antibody (validated for IP) at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate to immunoprecipitate PGM2L1 and analyze co-precipitating PMM1.

  • Perform reciprocal IPs with anti-PMM1 antibodies to confirm interaction.

  • Include appropriate controls (IgG control, lysates from PGM2L1-knockdown cells).

• Comparative expression analysis:

  • Quantify relative expression levels of PGM2L1 and PMM1 in diverse tissues using validated antibodies.

  • Based on published findings, consider that PMM2 is 6-7 fold better expressed than PMM1, while PGM2 is >2-fold better expressed than PGM2L1 in certain cellular contexts .

• Functional interplay assessment:

  • Investigate how PMM1 affects Glc-1,6-P2 levels:

    • Research has shown that transfection of PMM1 alone in cells almost depletes Glc-1,6-P2 (14-fold lower than control) .

    • When PMM1 is cotransfected with PGM2L1, it lowers Glc-1,6-P2 concentration to levels close to control values .

  • Compare with PMM2 effects, which only decrease Glc-1,6-P2 levels by approximately 25% when cotransfected with PGM2L1 .

• Knockdown/overexpression experiments:

  • Use siRNA knockdown of PGM2L1 to assess effects on PMM1 activity and vice versa.

  • Overexpress PGM2L1 in cellular models and measure changes in glucose metabolism parameters.

  • Based on published research, PGM2L1 overexpression significantly increases intracellular Glc-1,6-P2 levels compared to control cultures .

• Subcellular localization studies:

  • Perform immunofluorescence co-localization studies to determine whether PGM2L1 and PMM1 share subcellular compartments.

  • Use antibodies validated for immunofluorescence applications.

How can PGM2L1 antibodies be employed in proteogenomic approaches to identify membrane trafficking components?

For implementing PGM2L1 antibodies in proteogenomic approaches to identify membrane trafficking components, consider this comprehensive methodology:

• Multi-omics integration strategy:

  • Combine antibody-based proteomics with transcriptomic data to create a robust cross-validation framework.

  • Follow the approach used in proteogenomic analysis of antibody secretion, which successfully identified components involved in membrane trafficking .

  • Implement web-based bioresources for visualizing differential regulation of genes during cellular differentiation, focusing on coat proteins, tethers, and SNAREs potentially interacting with PGM2L1 .

• Subcellular fractionation combined with immunoprecipitation:

  • Fractionate cells to isolate membrane compartments (ER, Golgi, plasma membrane).

  • Use validated PGM2L1 antibodies for immunoprecipitation from these fractions.

  • Perform mass spectrometry analysis of co-precipitating proteins to identify novel membrane trafficking partners.

  • Validate findings using reciprocal IPs and co-localization studies.

• CRISPR-based screening approaches:

  • Develop CRISPR screens targeting membrane trafficking components.

  • Use PGM2L1 antibodies to assess changes in PGM2L1 localization or abundance as readouts.

  • Analyze hits to identify novel factors involved in PGM2L1 trafficking or function.

• Proximity labeling techniques:

  • Generate BioID or APEX2 fusion constructs with PGM2L1.

  • Use PGM2L1 antibodies to validate expression and localization of fusion proteins.

  • Identify proteins in proximity to PGM2L1 in different subcellular compartments.

  • Focus analysis on membrane trafficking components for functional validation.

• Cross-species comparative analysis:

  • Leverage the cross-species reactivity of available PGM2L1 antibodies (human, mouse, rat) .

  • Compare PGM2L1 interactions across species to identify evolutionarily conserved trafficking mechanisms.

What are the implications of AMPK-mediated phosphorylation on PGM2L1 function and how can antibody-based approaches help elucidate this regulation?

To investigate AMPK-mediated phosphorylation of PGM2L1 and its functional implications using antibody-based approaches, implement this research framework:

• Phosphorylation-specific detection strategy:

  • Leverage findings from related research showing that proteins like BAIAP2L1 can undergo AMPK-dependent phosphorylation resulting in band shifts .

  • Develop a Western blot approach using existing PGM2L1 antibodies to detect mobility shifts indicative of phosphorylation.

  • Compare lysates from cells treated with AMPK activators (e.g., AICAR, metformin) versus inhibitors (e.g., Compound C).

• Phospho-specific antibody development and validation:

  • Identify potential AMPK phosphorylation sites in PGM2L1 based on consensus motifs.

  • Generate phospho-specific antibodies against these sites.

  • Validate using in vitro kinase assays with recombinant AMPK and PGM2L1 protein.

  • Confirm specificity using phosphatase treatment and phospho-mutant PGM2L1 constructs.

• Functional impact assessment:

  • Use validated PGM2L1 antibodies to immunoprecipitate the protein from cells with activated or inhibited AMPK.

  • Measure enzymatic activity of immunoprecipitated PGM2L1 to determine how phosphorylation affects its glucose 1,6-bisphosphate synthase function.

  • Compare these findings with known regulatory mechanisms of related proteins.

• Subcellular localization analysis:

  • Examine whether AMPK-mediated phosphorylation alters PGM2L1 subcellular distribution using fractionation approaches followed by Western blotting.

  • Perform immunofluorescence studies using validated antibodies to visualize potential localization changes upon AMPK activation or inhibition.

• Protein-protein interaction dynamics:

  • Investigate whether phosphorylation affects PGM2L1 interactions with other metabolic enzymes or regulatory proteins.

  • Use co-immunoprecipitation with PGM2L1 antibodies under various AMPK activity conditions.

  • Consider that AMPK-dependent and independent phosphorylation events have been observed during cell detachment in other proteins like BAIAP2L1 , suggesting complex regulatory mechanisms.