Traditional Western Blotting Research Articles

Resolving the current controversy of use and reuse of housekeeping proteins in ageing research: Focus on saving people’s tax dollars

The use of housekeeping genes and proteins to normalize mRNA and protein levels in biomedical research has faced growing scrutiny. Researchers encounter challenges in determining the optimal frequency for running housekeeping proteins such as β-actin, Tubulin, and GAPDH for nuclear-encoded proteins, and Porin, HSP60, and TOM20 for mitochondrial proteins alongside experimental proteins. The regulation of these proteins varies with age, gender, disease progression, epitope nature, gel running conditions, and their reported sizes can differ among antibody suppliers. Additionally, anonymous readers have raised concerns about peer-reviewed and published articles, creating confusion and concern within the research and academic institutions. To clarify these matters, this minireview discusses the role of reference housekeeping proteins in Western blot analysis and outlines key considerations for their use as normalization controls. Instead of Western blotting of housekeeping proteins, staining of total proteins, using Amido Black and Coomassie Blue can be visualized the total protein content on a membrane. The reducing repeated Western blotting analysis of housekeeping proteins, will save resources, time and efforts and in turn increase the number of competitive grants from NIH and funding agencies. We also discussed the use of dot blots over traditional Western blots, when protein levels are low in rare tissues/specimens and cell lines. We sincerely hope that the facts, figures, and discussions presented in this article will clarify the current controversy regarding housekeeping protein(s) use, reuse, and functional aspects of housekeeping proteins. The contents presented in our article will be useful to students, scholars and researchers of all levels in cell biology, protein chemistry and mitochondrial research.

Abstract PO1-23-10: Multi-omics analysis identifies the mechanism in cross-resistance to endocrine therapy and CDK4/6 inhibitor in breast cancer

Abstract Background In advanced/metastatic breast cancer (MBC) or recurrent breast cancer on prior endocrine therapy (ET), selective estrogen receptor downregulator (SERD) of fulvestrant and CDK4/6i are the mainstream therapy regimen. In the clinical, fulvestrant combined with CDK4/6i is the second-line therapy for breast cancer (BC) that had progressed during previous endocrine therapy. The alternation of backbone from aromatase inhibitor or tamoxifen (endocrine sensitive) to fulvestrant (endocrine resistance) partially comes from the mechanism of ET resistance. Moreover, in the PALOMA-2 trial, approximately 30% of patients developed into recurrence after two years of CDK4/6i treatment, indicating that the ET backbone may affect CDK4/6i efficacy, bringing an emerging clinical issue of cross-resistance. However, whether the cross-resistance of ET would be more sensitive to CDK4/6i or impair the efficacy of CDK4/6i is still an ambiguous and heterogeneous issue. Also, in CDK4/6i resistance, whether the mechanism of ET resistance is associated with the dysregulation of the cell cycle or activation of other “bypass” signaling pathways is not fully understood. Methods We use a 3D and 2D culture of ET-sensitive cell S0.5, tamoxifen-resistant cell TAM, and fulvestrant-resistant cell 182R6 as our model. The 3D culture was performed by using the Tools 3d culture plus kit. Drug responses to fulvestrant and ribociclib were conducted by using CCK-8 assay. The potential target and pathway involved in the mechanisms of cross-resistance were analyzed by using multi-omics analysis of sequential window acquisition of all theoretical mass spectra (SWATH-MS), RNA-seq, micro-western array (MWA), traditional western blots, and open access database. Results In comparison with S0.5, the sensitivity to fulvestrant was indeed reduced in 182R6 cell; however, we found the sensitivity to ribociclib was also reduced in 182R6 cell, which was in contrast to the ribociclib-sensitive TAM cell. The different response to ribociclib between 182R6 and TAM, indicating that cross-resistance between fulvestrant and ribociclib existed in the model of 182R6 and TAM would be another control for 182R6 to find the target with or without cross-resistance. With multi-omics analysis of SWATH-MS, RNA-seq, MWA, western blots, and open access database, we found some unique or high expression of targets and pathways including EGFR, HER2, CDK6, CDK2, MAPK, and TNF-α pathways in cross-resistance R6 cell than in parental and TAM cell. We reconfirmed the above targets by 3D culture and found MAPK signaling pathway of p-ERK was not affected under ribociclib treatment, indicating the important role of the MAPK signaling pathway in ribociclib resistance. Conclusion To our knowledge, these results provide the first preliminary mechanism of cross-resistance between fulvestrant and ribociclib. We found fulvestrant resistant cell R6 exhibited the phenomenon of cross-resistance to ribociclib, which was in contrast to ribociclib-sensitive TAM cell. We also found some unique or high expression of target and pathway in cross-resistance R6 cells than in parental and TAM cells. In the future, with compound library screening and in vivo animal models, we expect to discover an FDA-approved drug or potential compound to overcome the cross-resistance between fulvestrant and ribociclib. Citation Format: Ming-Feng Hou, Chung-Liang Li, Chih-Po Chiang. Multi-omics analysis identifies the mechanism in cross-resistance to endocrine therapy and CDK4/6 inhibitor in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-10.

Quantification of AMPA receptor subunits and RNA editing-related proteins in the J20 mouse model of Alzheimer's disease by capillary western blotting.

Accurate modelling of molecular changes in Alzheimer's disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting. Here, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice. We observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD. Our findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD.

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30μm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6h, and thus this method provides several advantages over traditional western blotting.

MEK1-ERK1/2 signaling regulates the cardiomyocyte non-sarcomeric actin cytoskeletal network.

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic β- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width.NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic β-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.

A Combined Western and Bead-Based Multiplex Platform to Characterize Extracellular Vesicles.

In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 μg for all nine antibodies, compared with ∼10 μg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field.

Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting

Ubiquitination is a crucial cellular pathway enabling normal cellular functions. Abnormalities in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases. Efforts to screen and develop small molecule inhibitors targeting portions of the ubiquitination cascade require rapid and robust methods for detecting ubiquitination. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides one-second time resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously, and reagent concentrations are identical to those used by traditional western blot-based ubiquitination assays. The developed BLI ubiquitination assay is a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.

Abstract P2023: Mitogen-activated Protein Kinase-directed Modulation Of The Non-sarcomeric Actin Cytoskeleton In Cardiomyocytes

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the widening and lengthening of cardiomyocytes, respectively. Mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinases 1/2 (ERK1/2) are known to control this directional growth of cardiomyocytes, such that activated MEK1 causes concentric hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 MEK1 inhibitor, or control GFP adenovirus. The resulting data pinpointed over 2700 sites on 324 proteins as changing their phosphorylation status with modulation of this signaling cascade. Gene ontology analysis found that cytoskeletal-associated proteins were the largest subset of proteins with phosphorylation changes. Traditional and PhosTag western blots verified that many of these cytoskeletal proteins change expression as well as their phosphorylation status in NRVMs. In vivo experiments showed that cytoplasmic actin expression was upregulated in MEK1 transgenic or ERK1/2 gene-deleted hearts. Furthermore, MEK1 transgenic adult cardiomyocytes had enhanced cytoplasmic actin at lateral membranes. Palladin, an actin nucleating protein, changed both phosphorylation status and isoform expression in response to modulation of MEK1-ERK1/2 signaling. Adeno-associated viruses were used to overexpress different palladin isoforms. Immunostaining for these proteins showed that localization of palladin isoforms moved from the subcortical compartment to the sarcomere, suggesting these isoforms preferentially associate with either cytoskeletal or sarcomeric actin. Future studies will further examine the signaling roles that cytoplasmic actin may play in healthy and diseased cardiomyocytes and delineate preferential binding of actin associated proteins between cytoplasmic and sarcomeric actin in cardiomyocytes.

Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers.

3151 Background: KRAS, a GTPase that regulates cellular growth, is the most frequently mutated oncogene in non-small cell lung, pancreatic and colorectal cancers. Hyperactivation of KRAS in mutant alleles result in upregulated signaling within the MAPK pathway leading to aberrant cell proliferation. In addition to the pan- and mutant-specific KRAS inhibitors, its upstream GEF activator, SOS1, has emerged as an attractive therapeutic target given its ability to regulate the active, GTP-loaded state of KRAS and mitigate MAPK pathway feedback. Methods: Utilizing our PRODEGY platform, we developed bifunctional CRBN-SOS1 degraders for the treatment of patients with KRAS-mutated cancers, independent of mutation type. Results: Our degraders display CRBN- and proteasome-mediated SOS1 degradation with DC50s < 15nM in high-throughput (HiBiT) and traditional Western Blot assays in multiple mutant KRAS cell lines. SOS1 degradation resulted in the reduction of active KRAS, HRAS and MRAS levels when assayed by RAF-RBD pulldowns in MIA PaCa-2 (KRAS G12C) cells. Other downstream signaling markers (pERK and pS6) were also decreased upon SOS1 degradation in KRAS G12C, G12V, G12S and G13D cell lines in 3D Western Blot assays. Notably, the observed pERK and pS6 inhibition was driven by SOS1 degradation and not inhibition as decreases in signaling was no longer observed when SOS1 degraders were outcompeted with CRBN competitors. To assess the antiproliferative effects of our SOS1 degraders, we developed 3D proliferation assays in various KRAS mutant (G12A, G12C, G12D, G12V, G12S, G13C and G13D) as well as KRAS amplified cell lines and observed IC50s ranging from 0.5 - 70nM. Treatment of CRBN KO and SOS1 KO H358 and MIA PaCa-2 cell lines with our SOS1 degraders resulted in 100- to 400-fold IC50 values, validating that the functional activity observed is dependent on its targets. Consistent with our in vitro data, treatment of SOS1 degraders in MIA PaCa-2 xenografts resulted in > 90% degradation and led to significant tumor growth inhibition. Given the diversity of mutations and tumors as well as acquired drug resistance to allele-specific KRAS inhibitors, we sought to determine if coupling SOS1 degradation with other EGFR-RAS-MAPK inhibitors could produce more potent responses to maximize patient benefit. Strong synergistic antiproliferative effects were observed when SOS1 degraders were co-treated with KRAS inhibitors AMG510 (G12C) and MRTX1133 (G12D) in respective KRAS-mutant cell lines as well as with MEK inhibition and EGFR inhibition. Lastly, combination of AMG510 or Trametinib with SOS1 degraders yielded strong synergy and a greater anti-tumor response in MIA PaCa-2 xenografts relative to the individual agents themselves. Conclusions: Together, these results highlight the potential of SOS1 degraders alone and in combination as therapeutic options for a variety of KRAS-driven cancers.

Comparison of Automated and Traditional Western Blotting Methods

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses.

Label‐Free Fluorescence Turn‐On Detection of Histidine‐Tagged Proteins Based on Intramolecular Rigidification Induced Emission

AbstractHistidine tag (His‐tag) is a representative epitope tag, which has been widely used in the purification or immobilization of recombinant proteins. In this study, a novel probe CTPE‐IDA‐Ni2+ based on intramolecular rigidification induced emission for fluorescence detecting His‐tagged proteins is successfully developed and experimentally demonstrated. This detecting system is based on TPE derivative incorporated IDA‐Ni2+ moiety for real‐time detection of His‐tagged proteins. In the presence of His‐tagged proteins, the IDA‐Ni2+‐His‐tag complex was formed, which restricted the rotation of two phenyl rings linked with TPE and subsequently resulting in the fluorescence turn on. Results have shown that our probe could directly detect His‐tagged proteins with good selectivity and fast response within 10 min. In addition, we have proved that this system could be used in fluorescent labeling of His‐tagged protein instead of traditional Western blot assay with simple procedure, high quality and low background interference, as well as without aggregation‐caused quenching (ACQ) problems like some organic dyes.

Development of an Automated Capillary Immunoassay to Detect Prion Glycotypes in Creutzfeldt-Jakob Disease

Creutzfeldt-Jakob disease (CJD) comprises a group of transmissible neurodegenerative diseases with vast phenotypic diversity. Sporadic CJD heterogeneity is predominantly influenced by the genotype at codon 129 of the prion-encoding gene and the molecular weight of PrPSc fragments after protease digestion, resulting in a classification of 6 subtypes of CJD (MM1, MM2, MV1, MV2, VV1, and VV2). The majority of cases with CJD can be distinguished using this classification system. However, a number of reported CJD cases are phenotypically unique from others within their same subtype, such as variably protease-sensitive prionopathies, or exist as a mixture of subtypes within the same patient. Western blotting of brain tissue, along with the genotyping of codon 129 of the prion-encoding gene, is considered the “gold standard” for the biochemical characterization of CJD. Western blotting requires a significant amount of prion protein for detection, is labor-intensive, and is also associated with high interassay variability. In addition to these limitations, a growing body of research suggests that unique subtypes of CJD are often undetected or misdiagnosed using standard diagnostic western blotting protocols. Consequently, we successfully optimized and developed a capillary-based western assay using the JESS Simple Western (ProteinSimple) to detect and characterize prion proteins from patients with CJD. We found that this novel assay consistently differentiated CJD type 1 and type 2 cases with a limit of detection 10 to 100× higher than traditional western blotting. Cases with CJD in which type 1 and type 2 coexist within the same brain region can be detected using type 1-specific and type 2-specific antibodies, and we found that there was remarkable specificity for the detection of cases with variably protease-sensitive prionopathy. The assay presented displays outstanding sensitivity, allowing for the preservation of valuable samples and enhancing current detection methods.

Optimization of Capillary-Based Western Blotting for MYO7A.

Myosin VIIA (MYO7A)-associated Usher syndrome type 1B (USH1B) is a severe disorder that impacts the auditory, vestibular, and visual systems of affected patients. Due to the large size (~7.5kb) of the MYO7A coding sequence, we have designed a dual adeno-associated virus (AAV) vector-based approach for the treatment of USH1B-related vision loss. Due to the added complexity of dual-AAV gene therapy, careful attention must be paid to the protein products expressed following vector recombination. In order to improve the sensitivity and quantifiability of our immunoassays, we adapted our traditional western blot protocol for use with the Jess™ Simple Western System. Following several rounds of testing, we optimized our protocol for the detection of MYO7A in two of our most frequently used sample types, mouse eyes, and infected HEK293 cell lysates.

RF04 | PSUN233 Design of an Ultra-Stable Insulin/Glucagon Fusion Protein

Abstract Insulin replacement therapy is lifesaving for insulin-dependent diabetic patients, but it comes with an ever-present threat of hypoglycemia, which can eventually cause ischemic events, arrhythmias, neurological damage, coma, or even death. Consequently, the risk of hypoglycemia remains the most significant obstacle that prevents insulin-dependent patients from attaining adequate metabolic control. One approach to solve this issue involves the co-injection of insulin and glucagon at a fixed ratio, which has been shown to improve glycemic control with prevention of hypoglycemia. The co-administration of both hormones is based on two known physiological processes: first, insulin is a potent inhibitor of glucagon activity, especially under hyperglycemic conditions, and second, glucagon overpowers insulin and stimulates hepatic glucose production under hypoglycemic circumstances. In this work, we took this concept further into developing an insulin/glucagon fusion protein that will provide glycemic control (by the insulin portion) while preventing hypoglycemia (through the glucagon portion). Because both hormones are known to be unstable and form amyloid-like fibrils, our approach consists of using a fibrillation-resistant single-chain insulin (SCI) analog fused to a novel fibrillation-resistant glucagon analog. These molecules were independently synthesized by Solid-Phase Peptide Synthesis (SPPS) and linked together from the C-Terminus of the glucagon analog to the N-Terminus of the SCI analog by trypsin-mediated ligation. By themselves or when fused, these ultra-stable analogs didn't form fibrils after one month of agitation at 37°C in PBS (pH 7.4). By contrast, native insulin started fibrillation at 12.14 ± 3.65 hours, and native glucagon at time 0. The glucagon analog was designed to be less potent than the native molecule to allow unopposed insulin activity while in hyperglycemia but maintaining full agonism to prevent hypoglycemia. The in vitro glucagon activity was measured by cAMP production in HEK293 expressing the glucagon receptor. The glucagon analog was determined to be a full agonist with an EC50 of 125.7 nM, a ∼58-fold potency difference to the native molecule (2.175 nM). The fusion protein was tested in vitro and in vivo to determine the activities of both moieties when fused. The in vitro insulin activity of the fusion protein measured by traditional and in-cell western blot was comparable to the SCI alone. The in vitro glucagon activity of the fusion protein was also similar to the glucagon analog alone (EC50 151.2 nM), therefore validating our fusion approach. The in vivo insulin and glucagon activities are currently being tested by subcutaneous injection in diabetic (STZ) and normal rats. So far, both insulin and glucagon moieties have shown to be fully active, and hypoglycemia has not been observed in rats. Increasing doses studies are underway. We believe that this approach will allow the design of next-generation insulin treatments protected against hypoglycemia, ultra-stable, and amenable for multiplatform delivery. Presentation: Saturday, June 11, 2022 1:54 p.m. - 1:59 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Accelerating PROTAC drug discovery: Establishing a relationship between ubiquitination and target protein degradation

PROTACs have emerged as a new class of drugs that can target the “undruggable” proteome by hijacking the ubiquitin proteasome system. Despite PROTACs’ success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using “TUBEs” as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed “UbMax” - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.

The expression of nerve growth factor in healthy and inflamed equine chondrocytes analysed by capillary western immunoassay

Nerve Growth Factor (NGF) is a signalling molecule for pain and inflammation. NGF is increased in synovial fluid from osteoarthritic humans and animals, compared to healthy controls. Monoclonal antibody therapy directed against NGF has been approved to treat pain in osteoarthritic dogs but despite many years of trialling, therapy has not been approved for human use. One reason for this is that adverse reactions with rapidly progressing osteoarthritis has occurred in some individuals. More detailed knowledge of NGF expression in joints is needed. In this study, capillary-based Simple Western was used to analyse NGF in cultured equine chondrocytes. Chondrocytes were collected post mortem from three macroscopically healthy intercarpal joints and three intercarpal joints with mild osteoarthritic changes. The chondrocytes were expanded to passage one and seeded in chondrogenic medium to maintain the phenotype. On day four, cells were either stimulated with LPS or kept untreated in medium. All cells were harvested on day five. Wes analysis of lysates did not show mature NGF but two proforms, 40 and 45 kDa, were identified. Results were confirmed with western blot. The same proforms were expressed in chondrocytes from healthy and osteoarthritic joints. Acute inflammation induced by LPS stimulation did not change the forms of expressed NGF. Capillary Simple Western offers a sensitive and sample-sparing alternative to traditional western blot. However, confirmation of peaks is imperative in order to avoid misinterpretation of findings. In addition, in this case the method did not offer the possibility of quantification advertised by the manufacturers.

Capillary electrophoresis Western blot using inkjet transfer to membrane

Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 – 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, β-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.

Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue.

Lipedema is a multifaceted chronic fat disorder characterized by the bilateral and disproportionate accumulation of fat predominantly in the lower body regions of females. Research strongly supports that estrogen factors likely contribute to the pathophysiology of this disease. We aim to help demonstrate this link by quantifying estrogen factor differences between women with and without lipedema. For time and lipedema adipose tissue conservation, the Protein Simple WES machine will be utilized in place of traditional western blotting. Here, we are interested in evaluating estrogen related factors, such as, but not limited to, estrogen receptors and enzymes involved in the successive conversions of cholesterol and androgens to estrogens in human subcutaneous adipose. Evaluation of these factors within adipose tissue, however, is novel for this instrument. Thus, we optimized tissue lysis and protein extraction for 11 proteins of interest. Antibodies and their working concentrations were determined based upon specific and distinguishable (signal-to-noise) peaks from electropherogram outputs across different tissue lysate concentrations. We found that overnight acetone precipitation proved to be the best procedure for extracting protein from lipid rich adipose tissue samples. Six of the eleven proteins were found to migrate to their expected molecular weights, however, five did not. For proteins that did not migrate as expected, overexpression lysates and empty vector controls were used to validate detection antibodies. Protein extract from subcutaneous adipose tissue and overexpression lysates were then combined to understand if migration was specifically altered by adipose tissue. From these results, we concluded that the lipid rich nature of adipose tissue in combination with the separation matrix designated for use with the WES were preventing the appropriate migration of some proteins rather than non-specific antibody binding or inappropriate preparation methods.

Summit: Automated Analysis of Arrayed Single-Cell Gel Electrophoresis.

New pipelines are required to automate the quantitation of emerging high-throughput electrophoretic (EP) assessment of DNA damage, or proteoform expression in single cells. EP cytometry consists of thousands of Western blots performed on a microscope slide-sized gel microwell array for single cells. Thus, EP cytometry images pose an analysis challenge that blends requirements for accurate and reproducible analysis encountered for both standard Western blots and protein microarrays. Here, we introduce the Summit algorithm to automate array segmentation, peak background subtraction, and Gaussian fitting for EP cytometry. The data structure storage of parameters allows users to perform quality control on identically processed data, yielding a ~6.5% difference in coefficient of quartile variation (CQV) of protein peak area under the curve (AUC) distributions measured by four users. Further, inspired by investigations of background subtraction methods to reduce technical variation in protein microarray measurements, we aimed to understand the trade-offs between EP cytometry analysis throughput and variation. We found an 11%-50% increase in protein peaks that passed quality control with a subtraction method similar to microarray "average on-boundary" versus an axial subtraction method. The background subtraction method only mildly influences AUC CQV, which varies between 1% and 4.5%. Finally, we determined that the narrow confidence interval for peak location and peak width parameters from Gaussian fitting yield minimal uncertainty in protein sizing. The AUC CQV differed by only ~1%-2% when summed over the peak width bounds versus the 95% peak width confidence interval. We expect Summit to be broadly applicable to other arrayed EP separations, or traditional Western blot analysis.

Improved cell signaling analysis by biofunctionalized nanospheres and imaging flow cytometry.

The analysis of immune cell signaling is critical for the understanding of the biology and pathology of the immune system, and thus a mandatory step for the development of efficient biomarkers and targeted therapies. Phosflow, which has progressively replaced the traditional western blot approach, relies on flow cytometry to analyze various signaling pathways at a single-cell level. This technique however suffers a lack of sensitivity largely due to the low signal/noise ratio that characterizes cell signaling analysis. In this study, we describe a new technique, which combines the use of biofunctionalized nanospheres (i.e., synthetic particulate antigens, SPAg) to stimulate the immune cells in suspension and imaging flow cytometry to identify homogenously-stimulated cells and quantify the activity of the chosen signaling pathway in selected subcellular regions of interest. Using BCR signaling as model, we demonstrate that SIBERIAN (SPAg-assIsted suB-cEllulaR sIgnaling ANalysis) allows assessing immune cell signaling with unprecedented sensitivity and specificity.