PFKM Antibody

What is PFKM and why is it significant in research?

PFKM (phosphofructokinase, muscle) is a key regulatory enzyme in the glycolytic pathway that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate using ATP as a phosphate donor . This reaction represents one of the most important regulatory steps in glycolysis, making PFKM a critical target for metabolic research. The enzyme belongs to the phosphofructokinase family and specifically the two domains subfamily . The biochemical reaction catalyzed by PFKM can be represented as: ATP + D-fructose 6-phosphate = ADP + D-fructose 1,6-bisphosphate, highlighting its role in energy metabolism . Defects in PFKM are associated with glycogen storage disease type 7 (GSD7), also known as Tarui disease, making it relevant for both basic metabolic research and disease-oriented investigations .

How does PFKM differ from other phosphofructokinase isoforms?

PFKM is just one of several tissue-specific isoforms of phosphofructokinase expressed in mammals. While the search results don't provide comprehensive details about all isoforms, they do mention PFKL (liver isoform) and PFKP (platelet isoform) alongside PFKM (muscle isoform) . Each isoform exhibits tissue-specific expression patterns and potentially different regulatory properties. Research indicates that PFKM is predominantly expressed in skeletal muscle, heart tissue, and certain cell lines like HeLa and HepG2 . The tissue distribution of PFKM compared to other isoforms provides critical contextual information for researchers selecting appropriate experimental models. Additionally, the phosphorylation state of different PFK isoforms may play a role in metabolic reprogramming, as suggested by research on PFKL phosphorylation at Ser775 .

Which applications are commonly supported by commercial PFKM antibodies?

Commercial PFKM antibodies have been validated for multiple applications, with Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), and ELISA being the most common . According to published research, Western Blot applications are the most extensively validated, with 64 publications cited for one antibody product alone . The applications for which each antibody has been tested are typically listed in product information sheets, along with the specific reactivity observed in various sample types . For example, antibody 55028-1-AP has shown positive Western Blot detection in HeLa cells, human skeletal muscle tissue, HepG2 cells, human brain tissue, HEK-293 cells, mouse heart tissue, and rat heart tissue .

What are the recommended dilutions for various PFKM antibody applications?

Optimal dilutions for PFKM antibodies vary by application type and the specific antibody product. Based on the available information, the following dilution ranges are recommended:

What sample types have been validated for PFKM antibody detection?

PFKM antibodies have been validated across a variety of sample types, predominantly focusing on tissues and cell lines where PFKM is known to be expressed. According to the search results, positively validated sample types include:

• For Western Blot: HeLa cells, human skeletal muscle tissue, HepG2 cells, human brain tissue, HEK-293 cells, mouse heart tissue, rat heart tissue, PC-3 cells, and Raji cells .

• For Immunohistochemistry: Mouse and human skeletal muscle tissue samples, typically using paraffin-embedded sections .

• For Immunofluorescence/ICC: HeLa cells and HepG2 cells have been consistently validated .

• For Immunoprecipitation: Mouse heart tissue, mouse liver tissue, and mouse skeletal muscle tissue .
  Researchers should select sample types based on these validated examples to ensure reliable results, especially when establishing new experimental protocols.

Why might Western blot for PFKM show bands outside the expected molecular weight range?

PFKM typically appears at 75-85 kDa on Western blots, though its calculated molecular weight is around 85 kDa . Observing bands outside this range could be attributed to several factors. First, PFKM exists in multiple isoforms (85, 82, and 93 kDa) , which may present as distinct bands depending on the specificity of the antibody and the tissue being analyzed. Second, post-translational modifications like phosphorylation can alter protein migration patterns, as evidenced by studies on phosphorylated forms of PFKL . Third, degradation products or cross-reactivity with other phosphofructokinase family members might yield unexpected bands. To troubleshoot, researchers should include positive controls from validated tissues like skeletal muscle or heart tissue , use reducing conditions as employed in published protocols , and consider optimization of sample preparation methods to preserve protein integrity.

What are the most common technical issues with PFKM immunohistochemistry?

When performing immunohistochemistry for PFKM, several technical challenges may arise. First, antigen retrieval conditions are critical - recommended protocols suggest using TE buffer at pH 9.0, though citrate buffer at pH 6.0 can serve as an alternative . Insufficient or excessive antigen retrieval can significantly impact staining quality. Second, antibody dilution must be carefully optimized, with substantial variation between different antibody products (from 1:20 to 1:1600 depending on the specific antibody) . Third, tissue fixation methods can affect epitope accessibility - PFKM antibodies are typically validated on formalin-fixed paraffin-embedded tissues, so alternative fixation methods may require additional optimization . To address these issues, researchers should follow specific protocols provided by manufacturers, include positive control tissues (particularly skeletal muscle), and systematically optimize each step of the staining procedure.

How should PFKM antibodies be stored to maintain optimal activity?

Proper storage of PFKM antibodies is essential for maintaining their performance over time. According to multiple sources, PFKM antibodies should be stored at -20°C . Most PFKM antibodies are supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 , though some variations exist between manufacturers. These storage conditions typically ensure antibody stability for one year after shipment . Importantly, for -20°C storage, aliquoting is generally considered unnecessary according to manufacturer guidelines , which differs from common practice with many other antibodies. Some smaller volume preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizing agent . When using the antibody, it should be thawed completely and brought to room temperature before opening to prevent condensation that could introduce contamination or contribute to antibody degradation.

How can PFKM antibodies be used to investigate metabolic reprogramming in disease states?

PFKM antibodies provide powerful tools for investigating metabolic reprogramming in various pathological conditions. As a key regulatory enzyme in glycolysis, changes in PFKM expression or post-translational modification can indicate altered metabolic states in diseases such as cancer, diabetes, or muscular disorders. Recent research has demonstrated that phosphorylation of phosphofructokinase family members (specifically PFKL) plays a role in metabolic reprogramming, suggesting similar regulatory mechanisms might apply to PFKM . Researchers can employ PFKM antibodies in comparative studies between normal and disease tissues to quantify expression differences using Western blot or immunohistochemistry techniques . Moreover, the combination of PFKM antibodies with antibodies against other glycolytic enzymes can provide a more comprehensive view of metabolic pathway alterations. For instance, examining the ratio of PFKM to PFKL or PFKP expression using specific antibodies against each isoform could reveal tissue-specific metabolic adaptations in disease states .

What approaches can be used to simultaneously detect PFKM with other metabolic enzymes?

Multiplex detection of PFKM alongside other metabolic enzymes can provide valuable insights into glycolytic pathway regulation and metabolic network dynamics. Several approaches can be employed for this purpose. First, co-immunofluorescence staining using PFKM antibody in combination with antibodies against other glycolytic enzymes (with different host species to avoid cross-reactivity) allows visualization of spatial relationships between enzymes within cells or tissues . Second, sequential immunoprecipitation can be performed, where PFKM is immunoprecipitated first using validated IP protocols (0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate) , followed by Western blot analysis of the precipitate for interacting proteins. Third, for broader pathway analysis, researchers can employ a panel of antibodies against key glycolytic enzymes (including PFKM) in parallel Western blots of fractionated cell components to determine subcellular localization patterns under different metabolic conditions. When designing such experiments, careful consideration must be given to antibody compatibility, specifically regarding host species, detection methods, and potential cross-reactivity between closely related enzymes.

What are the considerations for using PFKM antibodies in studies of glycogen storage disease type 7 (GSD7)?

Glycogen storage disease type 7 (GSD7), also known as Tarui disease, results from defects in the PFKM gene . When investigating this disorder using PFKM antibodies, several specialized considerations apply. First, antibody selection should focus on products that can detect mutant forms of PFKM commonly found in GSD7 patients, which may have altered epitope accessibility or expression levels compared to wild-type protein. Second, tissue selection is critical - while skeletal muscle is the primary tissue affected in GSD7, comparative analysis with other tissues like heart and liver can provide insights into tissue-specific consequences of PFKM dysfunction . Third, when analyzing patient samples, appropriate controls must be included to distinguish between reduced expression and altered enzyme activity, which may not always correlate. Western blot analysis using validated PFKM antibodies (at dilutions of 1:2000-1:16000 for antibody 55028-1-AP or comparable dilutions for other products) can determine whether PFKM protein is absent, reduced, or present but non-functional in patient samples. Additionally, immunohistochemistry on muscle biopsies can reveal the distribution pattern of PFKM and potential accumulation of glycogen in affected tissues.

Which species have confirmed reactivity with available PFKM antibodies?

Commercial PFKM antibodies exhibit reactivity across multiple species, though the extent of validation varies between products. According to the search results, human, mouse, and rat species have been consistently confirmed to react with available PFKM antibodies . For instance, antibody 55028-1-AP has demonstrated positive Western blot results in human, mouse, and rat samples . Beyond these three main species, some antibodies have been cited as reactive with additional species including pig, chicken, bovine, and sheep, though these appear to have less extensive validation data . The monoclonal antibody MAB7687 specifically claims reactivity with human, mouse, and rat muscle phosphofructokinase as demonstrated through Western blot analysis of heart tissue from all three species . When selecting an antibody for cross-species studies, researchers should prioritize products with explicit validation data for each species of interest rather than relying on predicted cross-reactivity.

How conserved is PFKM across different species, and how does this affect antibody selection?

The conservation of PFKM across species impacts antibody epitope recognition and consequently affects antibody selection for cross-species studies. While the search results don't provide specific sequence homology data, the demonstrated cross-reactivity of multiple antibodies with human, mouse, and rat PFKM suggests substantial conservation of epitopes among mammalian species . Some antibodies are raised against specific peptide regions, such as the C-terminal portion (for instance, the antibody described in result #5 targets human PFKM amino acids Asn674-Val780) , while others use fusion proteins as immunogens . When selecting antibodies for novel species applications, researchers should consider whether the immunogen sequence is conserved in their target species. Additionally, epitope accessibility may vary between species due to differences in post-translational modifications or protein-protein interactions, potentially affecting antibody performance even when the primary sequence is conserved. Preliminary validation experiments, such as Western blot analysis with positive control samples from the species of interest, are strongly recommended before proceeding with large-scale studies using PFKM antibodies in non-validated species.

What validation steps should be taken when using PFKM antibodies in non-validated species?

When extending PFKM antibody use to species not explicitly validated by manufacturers, researchers should implement a systematic validation approach. First, perform bioinformatic analysis to assess sequence homology between the validated species and the target species, particularly focusing on the immunogen region if known . Higher sequence conservation increases the probability of cross-reactivity. Second, conduct Western blot validation using tissues known to express high levels of PFKM in the target species (typically skeletal muscle or heart tissue) . This should include appropriate positive controls from validated species run in parallel. Third, verify specificity through complementary approaches such as siRNA knockdown or comparison with recombinant protein standards when possible. Fourth, optimize experimental conditions specifically for the new species, including sample preparation, antibody dilution, incubation times, and detection methods. Finally, consider validating results with an alternative PFKM antibody recognizing a different epitope to confirm findings. These validation steps are essential to ensure reliable and reproducible results when extending PFKM antibody applications beyond manufacturer-validated species.

What positive and negative controls should be included in PFKM antibody experiments?

Proper controls are essential for reliable interpretation of PFKM antibody experiments. For positive controls, tissues or cell lines with well-documented PFKM expression should be included. Based on the search results, recommended positive controls include: human or mouse skeletal muscle tissue for Western blot, IHC, and IP applications ; HeLa cells or HepG2 cells for Western blot and immunofluorescence studies ; and heart tissue from human, mouse, or rat for Western blot applications . For negative controls, several approaches should be considered: primary antibody omission controls to assess non-specific binding of secondary detection reagents; isotype controls using non-specific IgG from the same host species as the PFKM antibody at equivalent concentration; and when possible, PFKM-depleted samples (through siRNA knockdown or from PFKM-deficient models). Additionally, peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide before application to the sample, can confirm binding specificity. Including these controls systematically will enhance the reliability and interpretability of results obtained with PFKM antibodies.

How should experimental conditions be optimized for different sample types?

Optimization of experimental conditions for PFKM antibody applications varies significantly depending on sample type and detection method. For tissue samples in Western blot applications, proper homogenization and protein extraction buffers are critical - protocols using reducing conditions have been successfully employed . For cell lines, standard lysis buffers containing protease inhibitors are typically sufficient . For immunohistochemistry applications, antigen retrieval represents a critical step - TE buffer at pH 9.0 is recommended as the primary method, with citrate buffer at pH 6.0 as an alternative . The dilution of primary antibody requires careful titration, with significant variation between different antibodies and applications (from 1:10 for immunofluorescence to 1:50000 for Western blot) . Incubation conditions (time, temperature) should also be optimized - for instance, Western blot protocols for PFKM antibody 84281-5 have specific recommendations that may differ from standard protocols . Sample-dependent variables, such as protein loading amounts, blocking reagents, and detection methods, should be systematically tested to establish optimal conditions for each experimental system.

What approaches can detect post-translational modifications of PFKM?

Detection of post-translational modifications (PTMs) of PFKM requires specialized antibodies and techniques. While the search results don't provide specific antibodies against modified PFKM, they do mention research on phosphorylated PFKL, where investigators raised antibodies against phosphorylated and unphosphorylated peptides containing Ser775 . A similar approach could be applied to PFKM, developing modification-specific antibodies that recognize phosphorylated, acetylated, or otherwise modified forms of the protein. To detect PTMs of PFKM, researchers can employ several strategies: first, immunoprecipitation using total PFKM antibodies followed by Western blot with PTM-specific antibodies (e.g., anti-phosphoserine); second, two-dimensional gel electrophoresis to separate PFKM isoforms based on charge (affected by phosphorylation) followed by Western blot; third, mass spectrometry analysis of immunoprecipitated PFKM to identify and quantify specific modifications. When designing such experiments, researchers should consider the dynamic nature of PTMs and incorporate appropriate controls for modification status, such as samples treated with phosphatases or deacetylases to remove the modifications of interest.

How should quantitative differences in PFKM expression be analyzed across experimental conditions?

Quantitative analysis of PFKM expression requires careful consideration of several methodological factors. For Western blot applications, densitometric analysis should be performed on bands within the 75-85 kDa range, which corresponds to the observed molecular weight of PFKM . Normalization is critical - for total protein expression, housekeeping proteins such as GAPDH, β-actin, or tubulin can be used, though researchers should verify that these reference proteins are not affected by experimental conditions. Alternatively, total protein stains like Ponceau S provide a loading control less susceptible to experimental variation. When comparing PFKM expression across tissues, normalization strategies may need adjustment since housekeeping gene expression can vary between tissue types. For immunohistochemistry or immunofluorescence quantification, both staining intensity and the percentage of positive cells/area should be considered. Digital image analysis using software that can distinguish between specific staining and background is recommended. Statistical analysis should include appropriate tests for the experimental design (t-tests for two-group comparisons, ANOVA for multiple groups, etc.) with consideration of data distribution and variance.

How can contradictory results between different PFKM antibody applications be reconciled?

Contradictory results between different detection methods using PFKM antibodies (e.g., positive Western blot but negative IHC) can occur for various reasons. First, epitope accessibility differs between applications - denatured proteins in Western blot versus partially preserved structure in IHC or IP may affect antibody binding . Second, sensitivity thresholds vary across methods, with Western blot typically offering higher sensitivity than IHC. Third, fixation procedures for IHC or permeabilization for IF can alter epitope recognition. To reconcile contradictory results, researchers should: verify antibody performance using positive controls for each application; ensure that application-specific protocols are optimized (using recommended dilutions for each method) ; consider using alternative antibodies that recognize different epitopes of PFKM; and evaluate whether post-translational modifications might affect epitope recognition in different applications. When reporting contradictory results, full transparency about methodological details is essential, including antibody catalog numbers, dilutions, incubation conditions, and detection methods for each application.

What strategies can differentiate between PFKM isoforms in experimental settings?

Differentiating between the three reported isoforms of PFKM (85, 82, and 93 kDa) requires specialized experimental approaches. First, high-resolution SDS-PAGE with extended running times can physically separate these closely sized isoforms, particularly when using gradient gels (e.g., 6-12%). Second, isoform-specific antibodies could be developed targeting unique regions of each isoform, though the search results don't indicate commercial availability of such antibodies. Third, two-dimensional electrophoresis (separating by both isoelectric point and molecular weight) can distinguish isoforms that may have different post-translational modification patterns. Fourth, RT-PCR using primers specific to each isoform can determine their relative expression at the mRNA level, providing complementary data to protein analysis. Fifth, mass spectrometry following immunoprecipitation with a pan-PFKM antibody can identify peptides unique to each isoform. When analyzing complex samples that may contain multiple PFKM isoforms, researchers should clearly report the molecular weight of the band(s) observed and avoid making claims about specific isoforms unless validated methods for their distinction have been employed.