AKT2 Antibody

What is AKT2 and what cellular functions does it regulate?

AKT2 is a serine/threonine kinase in the AKT family, which includes three closely related members: AKT1, AKT2, and AKT3. These enzymes are collectively known as AKT kinase and regulate numerous cellular processes including metabolism, proliferation, cell survival, growth, and angiogenesis through phosphorylation of downstream substrates . Over 100 substrates have been reported thus far, though in many cases the specific AKT isoform responsible for the phosphorylation events remains uncharacterized .

AKT2 plays a particularly important role in the insulin signaling pathway and glucose metabolism. It regulates glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface . AKT2 is required to induce glucose transport and is therefore critical for maintaining metabolic homeostasis . Additionally, AKT2 contributes to glycogen synthesis by phosphorylating GSK3 isoforms, resulting in inhibition of their kinase activity .

What are the recommended experimental applications for AKT2 antibodies?

AKT2 antibodies can be utilized in multiple experimental applications, with effectiveness varying by specific antibody clone and manufacturer. Based on validation data from multiple sources, common applications include:

These applications have been validated on human and mouse samples, with cross-reactivity reported for rat samples in some antibodies . It is recommended to titrate each antibody in your specific system to obtain optimal results, as sample type can influence performance .

How should I store and handle AKT2 antibodies to maintain their activity?

Proper storage and handling of AKT2 antibodies is crucial for maintaining their specificity and activity. Most commercially available AKT2 antibodies should be stored at -20°C for long-term preservation . For short-term storage (up to two weeks), refrigeration at 2-8°C is generally acceptable .

Many AKT2 antibodies are supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This formulation helps maintain antibody stability during freeze-thaw cycles. Nevertheless, it is advisable to aliquot the antibody upon receipt to minimize repeated freeze-thaw cycles that can degrade antibody quality .

When working with the antibody, avoid contamination and maintain sterile technique. Always centrifuge the antibody vial before opening to ensure all liquid is at the bottom of the vial. For antibodies containing BSA (such as some 20μl size preparations), no further aliquoting is typically necessary for -20°C storage .

How can I distinguish between AKT isoforms in my experiments?

Distinguishing between AKT1, AKT2, and AKT3 in experimental settings requires careful selection of antibodies and methodological approaches due to the high sequence homology between these isoforms. Here are key strategies:

First, select isoform-specific antibodies that have been validated for specificity. Some AKT2 antibodies are developed using immunogens corresponding to unique regions of AKT2, such as the C-terminal region (amino acids 400 to C-terminus) that differs from other isoforms . Look for antibodies specifically tested for lack of cross-reactivity with other AKT family members. For example, certain commercially available antibodies detect "endogenous levels of Akt2 proteins in normal cell lysates without cross-reactivity with other family members" .

When performing Western blot analysis, slight molecular weight differences can sometimes be observed (AKT2 typically runs at 56-60 kDa) . Additionally, using positive and negative controls is essential—cell lines or tissues known to differentially express AKT isoforms can help confirm antibody specificity.

For functional studies, isoform-specific knockdown approaches using shRNA or siRNA targeting unique regions of AKT2 can confirm antibody specificity and isoform-specific functions .

What is the role of AKT2 in cancer metastasis and how can it be studied?

AKT2 plays a significant role in cancer progression, particularly in metastasis. Research has demonstrated that AKT2 deficiency profoundly inhibits the development of liver lesions in mouse models of colorectal cancer, whereas AKT1 deficiency had no effect under the same experimental conditions . This suggests an isoform-specific function in metastatic progression.

To study AKT2's role in metastasis, researchers can employ several methodological approaches:

• Knockdown/knockout studies: Targeted knockdown of AKT2 using shRNA or CRISPR-Cas9 approaches can reveal the impact of AKT2 deficiency on metastatic potential. For example, studies have shown that Akt2 knockdown resulted in only 35% of animals developing liver metastases compared to control groups .

• Metastasis suppressor analysis: AKT2 has been linked to regulation of metastasis suppressor 1 (MTSS1) expression. Immunoblot analysis can reveal that knockdown of AKT2, but not AKT1 or AKT3, robustly increases MTSS1 levels . This regulatory relationship can be further studied through downstream components of the MTSS1-Src-CTTN inhibitory axis, as AKT2 knockdown markedly decreases levels of active pSrc (Y416) and pCTTN (Y421) .

• In vivo metastasis models: Experimental metastasis assays using fluorescently labeled cancer cells allow for quantification of metastatic burden by analyzing expression of human markers (e.g., GAPDH) in mouse tissues or by fluorescence microscopy to count metastatic foci .

• Cell migration assays: Transwell migration assays reveal that AKT2 knockdown can impair cancer cell migration, with studies showing approximately 70% reduction in motility at 36 hours post-seeding .

• Cell viability and survival analysis: MTT assays and DNA fragmentation analysis can assess the impact of AKT2 deficiency on cancer cell viability and apoptosis, important components of the metastatic cascade .

How can I detect activation status of AKT2 in response to insulin and nutrient signaling?

Detecting the activation status of AKT2 in response to insulin and nutrient signaling requires methods that can specifically assess AKT2 phosphorylation and its downstream effects. AKT2 is a key mediator in the insulin signaling pathway, regulating glucose uptake and metabolism .

For direct detection of AKT2 activation:

• Phospho-specific antibodies: Use antibodies that recognize phosphorylated AKT2 at key regulatory sites, particularly Thr309 and Ser474, which are phosphorylated upon activation. Western blotting with these antibodies following insulin stimulation can reveal activation kinetics.

• Immunoprecipitation followed by Western blotting: Immunoprecipitate total AKT2 using isoform-specific antibodies, then probe with phospho-specific antibodies to determine the proportion of activated AKT2.

• In vitro kinase assays: After immunoprecipitating AKT2, assess its kinase activity using appropriate substrates and measuring phosphorylation levels.

For functional readouts of AKT2 activation:

• GLUT4 translocation assays: Since AKT2 regulates insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface, assessing membrane localization of GLUT4 provides a functional readout of AKT2 activity .

• Downstream substrate phosphorylation: Monitor phosphorylation status of AKT2-specific substrates. For example, AKT2 phosphorylates PTPN1 at 'Ser-50', which negatively modulates its phosphatase activity and prevents dephosphorylation of the insulin receptor . Similarly, TBC1D4 phosphorylation by AKT2 triggers binding of inhibitory 14-3-3 proteins, which is required for insulin-stimulated glucose transport .

• Glycogen synthesis: Since AKT2 regulates glycogen storage through GSK3 phosphorylation, measuring glycogen synthesis rates following insulin stimulation can provide an indirect measure of AKT2 activity .

What are common challenges in AKT2 detection and how can they be addressed?

Researchers frequently encounter several challenges when working with AKT2 antibodies in various applications. Here are common issues and their solutions:

Issue: Weak or no signal in Western blotting

• Solution: Optimize protein extraction by using buffers containing phosphatase inhibitors, especially when detecting phosphorylated AKT2. Cell lysis buffers containing NP-40 or RIPA with protease and phosphatase inhibitor cocktails are recommended. Additionally, adjust antibody concentration following manufacturer recommendations (typically 1:500-1:1000 for Western blots) .

Issue: Non-specific bands

• Solution: Increase blocking time (5% BSA or milk is typically effective), optimize antibody concentration, and include appropriate negative controls. For AKT2 detection, which typically shows a band at 56-60 kDa, it's important to verify specificity using Akt2 knockout/knockdown samples .

Issue: Poor reproducibility in immunohistochemistry

• Solution: Standardize antigen retrieval methods. For some AKT2 antibodies, TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may be used alternatively . Optimize antibody dilution (1:50-1:500 range) and incubation conditions based on tissue type .

Issue: Distinguishing AKT2 from other AKT family members

• Solution: Select antibodies specifically validated for lack of cross-reactivity with AKT1 and AKT3. Some manufacturers provide antibodies that "detect endogenous levels of Akt2 proteins in normal cell lysates without cross-reactivity with other family members" .

Issue: Variable results across different cell/tissue types

• Solution: Sample-dependent optimization is crucial. Test antibody performance in your specific model system before conducting full experiments. Different tissue types may require different dilutions or incubation conditions .

How can I optimize detection of AKT2 in different cellular compartments?

AKT2 localization can vary depending on activation status and cellular context. To optimize detection in different cellular compartments, consider these methodological approaches:

• Subcellular fractionation: Prior to Western blotting, perform subcellular fractionation to isolate cytoplasmic, membrane, nuclear, and cytoskeletal fractions. This allows for assessment of AKT2 distribution across cellular compartments. Use compartment-specific markers (e.g., GAPDH for cytoplasm, Na⁺/K⁺ ATPase for plasma membrane) as controls.

• Immunofluorescence optimization:

  • Fixation: 4% paraformaldehyde preserves most epitopes while maintaining cellular architecture

  • Permeabilization: Titrate detergent concentration (e.g., 0.1-0.5% Triton X-100) to ensure access to intracellular AKT2 without disrupting membrane structures

  • Antibody dilution: Test a range within 1:50-1:500 as recommended by manufacturers

  • Include co-staining with compartment-specific markers (e.g., DAPI for nucleus, phalloidin for actin cytoskeleton)

• Confocal microscopy: Use z-stack imaging to precisely locate AKT2 within three-dimensional cellular space, particularly when assessing membrane translocation following insulin stimulation.

• Stimulus-dependent localization: When studying AKT2 translocation in response to insulin or other stimuli, optimize time-course experiments to capture dynamic localization changes. Fixed timepoints (e.g., 5, 15, 30 min) following stimulation can reveal the kinetics of redistribution.

• Proximity ligation assay (PLA): For detecting interactions of AKT2 with other proteins in specific compartments, PLA provides spatial resolution of protein-protein interactions within intact cells.

How does AKT2 function differ from other AKT isoforms in cancer progression?

AKT isoforms exhibit distinct functions in cancer progression despite their structural similarities. Understanding these differences is crucial for targeting specific isoforms in cancer therapy. Research has revealed several key distinctions between AKT2 and other AKT family members in cancer:

AKT2 demonstrates a particularly important role in metastasis compared to AKT1. In colorectal cancer models, knockdown of Akt2 significantly reduced metastatic burden and the development of liver lesions, whereas Akt1 knockdown had no effect under the same experimental conditions . Quantification of metastatic spots in mouse livers revealed a significant reduction in the number of lesions per metastasis-bearing mouse upon loss of Akt2 (P = 0.01), but not Akt1 (P = 0.98) .

At the molecular level, AKT2 selectively regulates metastasis suppressor 1 (MTSS1) expression. Immunoblot analysis showed that knockdown of Akt2, but not Akt1 or Akt3, markedly induced expression of MTSS1 both in vitro and in vivo . This regulation affects downstream components of the MTSS1-Src-CTTN inhibitory axis, as Akt2 knockdown decreased levels of active pSrc (Y416) and pCTTN (Y421), whereas Akt1 or Akt3 knockdown had no effect .

Functionally, AKT2 deficiency impacts multiple aspects of the metastatic cascade. In colorectal cancer cells, Akt2 knockdown results in:

• Decreased cell viability as measured by MTT assays

• Approximately six-fold increase in apoptosis as determined by DNA fragmentation assays

• ~70% reduction in cell motility in transwell migration assays

• Reduced Ki67 staining in tumors, indicating decreased proliferation in vivo

• Increased TUNEL staining, demonstrating enhanced apoptosis in vivo

These findings suggest that while AKT1 is often associated with primary tumor growth, AKT2 may play a more prominent role in enabling metastatic spread, highlighting the importance of isoform-specific targeting in cancer therapeutics.

What methodological approaches can be used to study AKT2's role in glucose metabolism?

AKT2 plays a crucial role in glucose metabolism, particularly through its effects on insulin signaling. To study these functions, researchers can employ several methodological approaches:

• Glucose uptake assays: Given AKT2's role in mediating insulin-induced translocation of GLUT4 glucose transporters to the cell surface , researchers can use fluorescent glucose analogs (e.g., 2-NBDG) or radiolabeled glucose to measure uptake in cells with manipulated AKT2 expression or activity. Comparing wild-type, AKT2-overexpressing, and AKT2-deficient cells can reveal the quantitative contribution of AKT2 to glucose transport.

• GLUT4 translocation assays: Since AKT2 regulates glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface , researchers can use GLUT4 tagged with fluorescent proteins or measure surface GLUT4 levels by biotinylation assays to directly assess AKT2's functional impact.

• Insulin signaling pathway analysis: Examine phosphorylation of AKT2 substrates involved in glucose metabolism, including:

  • Phosphorylation of PTPN1 at 'Ser-50', which prevents dephosphorylation of the insulin receptor

  • Phosphorylation of TBC1D4, which triggers binding of inhibitory 14-3-3 proteins required for insulin-stimulated glucose transport

  • Phosphorylation of GSK3A at 'Ser-21' and GSK3B at 'Ser-9', which inhibits their kinase activity and promotes glycogen synthesis

• Glycogen synthesis assays: Since AKT2 regulates glycogen storage through phosphorylation of GSK3 isoforms , measuring glycogen content using methods such as periodic acid-Schiff staining or biochemical glycogen assays can provide insights into AKT2's metabolic functions.

• Metabolic phenotyping of AKT2 knockout/knockdown models: Assessing insulin sensitivity, glucose tolerance, and energy homeostasis in models with altered AKT2 expression. This can include:

  • Glucose tolerance tests

  • Insulin tolerance tests

  • Hyperinsulinemic-euglycemic clamps

  • Metabolic cage studies to measure energy expenditure

• Tissue-specific approaches: Since AKT2's metabolic effects may differ across tissues, conducting tissue-specific knockdown/knockout studies in key metabolic tissues (liver, muscle, adipose tissue) can reveal tissue-specific functions.

How can AKT2 antibodies be used to investigate AKT2's interactions with nutrient-sensing pathways?

AKT2 integrates into nutrient-sensing pathways and helps regulate energy status in response to external stimuli . Investigating these interactions requires specialized methodological approaches using AKT2 antibodies:

• Co-immunoprecipitation (Co-IP) studies: Using AKT2-specific antibodies for immunoprecipitation (recommended dilution 1:50) , researchers can pull down AKT2 complexes and identify interacting partners involved in nutrient sensing through subsequent Western blotting or mass spectrometry. This approach can reveal physical interactions between AKT2 and components of nutrient-sensing pathways such as mTOR complexes.

• Proximity ligation assay (PLA): This technique allows visualization of protein-protein interactions in fixed cells or tissues with single-molecule resolution. By using an AKT2-specific antibody alongside antibodies against putative interaction partners in nutrient-sensing pathways, researchers can detect and quantify specific interactions under different nutritional conditions.

• Chromatin immunoprecipitation (ChIP): For studying potential roles of AKT2 in transcriptional regulation of nutrient-responsive genes, ChIP using AKT2 antibodies can identify genomic regions bound by AKT2 or its complexes, particularly following nutrient stimulation or deprivation.

• Phospho-specific antibody approaches: Since AKT2 functions largely through phosphorylation of downstream targets, using phospho-specific antibodies against AKT2 substrates can map the signaling cascade in response to nutrient availability. Key targets include:

  • TSC2 at 'Ser-939' and 'Thr-1462', which activates mTORC1 signaling

  • 4E-BP1 and RPS6KB1, which are influenced by AKT2-mediated mTORC1 activation

  • FOXO transcription factors, which are phosphorylated by AKT2 leading to their cytoplasmic localization and inhibition

• Immunofluorescence microscopy: Using AKT2 antibodies (recommended dilution 1:50-1:500) for immunofluorescence can reveal the subcellular localization of AKT2 under different nutritional states, particularly in relation to nutrient-sensing organelles such as lysosomes (mTOR localization sites).

• Nutrient response time-course experiments: By treating cells with nutrients (e.g., amino acids, glucose) or nutrient-depleted media and analyzing AKT2 activation and localization at various time points, researchers can determine the temporal dynamics of AKT2's involvement in nutrient sensing.

What emerging techniques might enhance AKT2 isoform-specific research?

As research on AKT2 continues to evolve, several emerging techniques show promise for enhancing isoform-specific investigations:

• CRISPR-Cas9 genome editing: While RNAi approaches have been valuable for studying AKT2 function , CRISPR-Cas9 offers more complete and stable knockout of AKT2. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems additionally allow for endogenous gene modulation without complete knockout, enabling more nuanced study of AKT2 dosage effects.

• Optogenetic and chemogenetic tools: Development of AKT2-specific optogenetic constructs would allow for temporal and spatial control of AKT2 activity in living cells and organisms. This could reveal acute effects of AKT2 activation/inhibition with unprecedented precision.

• PROTAC (Proteolysis Targeting Chimera) technology: PROTAC molecules targeting AKT2 specifically could enable rapid, reversible, and complete degradation of endogenous AKT2 protein, offering advantages over genetic knockout approaches, particularly for studying acute responses.

• Single-cell phosphoproteomics: Advances in mass spectrometry sensitivity now enable phosphoproteomic analysis at the single-cell level, which could reveal cell-to-cell variation in AKT2 signaling networks within heterogeneous tissues.

• Spatial transcriptomics and proteomics: These techniques allow for spatial mapping of gene expression and protein abundance within tissues, which could reveal location-specific functions of AKT2 in complex tissues such as tumors or metabolic organs.

• Cryo-electron microscopy: Structural studies of AKT2 in complex with its binding partners could reveal isoform-specific interaction surfaces that might be targeted for therapeutic development.

• Biosensors for real-time AKT2 activity: Development of AKT2-specific FRET-based or other biosensors could enable real-time visualization of AKT2 activity in living cells, providing insights into the dynamics of activation and inhibition.

• Organoid models: Patient-derived organoids with manipulated AKT2 expression could provide more physiologically relevant models for studying AKT2 function in human tissues, particularly in disease contexts.

These emerging techniques, when combined with isoform-specific antibodies, hold promise for advancing our understanding of AKT2's unique functions in health and disease, potentially leading to more targeted therapeutic approaches.