A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Eaton, Samantha L.; Hurtado, Maica Llavero; Oldknow, Karla J.; Graham, Laura C.; Marchant, Thomas W.; Gillingwater, Thomas H.; Farquharson, Colin; Wishart, Thomas M.

doi:10.3791/52099-v

Cited by 5 publications

(7 citation statements)

References 5 publications

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“…SDS-PAGE was performed following the standard protocols. (Eaton et al 2014 ) Protein concentration was calibrated to 1 μg/μL with a 5 × protein loading bufer (Beyotime Biotechnology). Ten microliters of samples were loaded into each well after boiling for 10 min, and 4–20% non-reducing sodium dodecyl sulfat-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at 120 V for about 70 min.…”

Section: Methodsmentioning

confidence: 99%

Preclinical evaluation of the theranostic potential of 89Zr/177Lu-labeled anti-TROP-2 antibody in triple-negative breast cancer model

Wu,

li,

Zhang

et al. 2024

EJNMMI radiopharm. chem.

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Background Triple-negative breast cancer (TNBC) is one of the most lethal malignant tumors among women, characterized by high invasiveness, high heterogeneity, and lack of specific therapeutic targets such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Trophoblast cell-surface antigen-2 (TROP-2) is a transmembrane glycoprotein over-expressed in 80% of TNBC patients and is associated with the occurrence, progress, and poor prognosis of TNBC. The TROP-2 targeted immunoPET imaging allows non-invasive quantification of the TROP-2 expression levels of tumors, which could help to screen beneficiaries most likely to respond to SG and predict the response. This study aimed to develop a 89Zr/177Lu-radiolabeled anti-TROP-2 antibody (NY003) for immunoPET and SPECT imaging, as well as radioimmunotherapy (RIT) in TROP-2 (+)TNBC tumor-bearing model. Based on the camelid antibody, we developed a TROP-2 targeted recombinant antibody NY003. NY003 was conjugated with DFO and DTPA for 89Zr and 177Lu radiolabelling, respectively. The theranostic potential of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 was evaluated through immunoPET, SPECT imaging, and RIT studies in the subcutaneous TROP-2 positive TNBC xenograft mice model. Results The high binding affinity of NY003 to TROP-2 was verified through ELISA. The radiochemical purity of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 exceeded 95% and remained stable within 144h p.i. in vitro. ImmunoPET and SPECT imaging showed the specific accumulation of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 in MDA-MB-231 tumors and gradually increased with the time tested, significantly higher than that in control groups (P < 0.05). The strongest anti-tumor efficacy was observed in the high-dose of [177Lu]Lu-DTPA-NY003 group, followed by the low-dose group, the tumor growth was significantly suppressed by [177Lu]Lu-DTPA-NY003, the tumor volumes of both high- and low-dose groups were smaller than the control groups (P < 0.05). Ex vivo biodistribution and histological staining verified the results of in vivo imaging and RIT studies. Conclusion As a drug platform for radiotheranostics, 89Zr/177Lu-radiolabeled anti-TROP-2 antibody NY003 could not only non-invasively screen the potential beneficiaries for optimizing SG ADC treatment but also suppressed the growth of TROP-2 positive TNBC tumors, strongly supporting the theranostic potential of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003.

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Section: Methodsmentioning

confidence: 99%

Preclinical evaluation of the theranostic potential of 89Zr/177Lu-labeled anti-TROP-2 antibody in triple-negative breast cancer model

Wu,

li,

Zhang

et al. 2024

EJNMMI radiopharm. chem.

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“…Western blot analysis necessitates careful consideration of loading controls and signal linearity for accurate quantitative interpretation. While housekeeping proteins like GAPDH and actin remain widely used as loading controls, their expression levels can fluctuate across experimental conditions (23,24,15). In the case of FIGURE 1, while loading controls appear similar within each signaling time course, WT and mutant CXCR4 (S338/39A and S324/25A) samples are different.…”

Section: Examining Loading Controls and Signal Saturation In Western ...mentioning

confidence: 99%

“…This highlights the importance of confirming that the chosen loading control remains stable under your specific experimental treatments. Ideally, total protein staining should be used to normalize protein loading (25)(26)(27)15). This practice offers a more reliable representation of overall protein abundance.…”

Section: Examining Loading Controls and Signal Saturation In Western ...mentioning

confidence: 99%

“…In the following sections, we discuss how loading controls, experimental timescales, antibody stripping, and antibody banding patterns can shape Western blot interpretation. This perspective does not aim to provide a comprehensive technical review of Western blotting; several excellent articles offer in-depth guidance on best practice (14)(15)(16)(17)(18)(19)(20)(21)(22). Instead, we focus on assumptions and aspects of Western blotting that should be considered by both theorists and experimentalists when evaluating data.…”

Section: Introductionmentioning

confidence: 99%

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Seeing Beyond the Blot: A Critical Look at Assumptions and Raw Data Interpretation in Western Blotting

DeNies,

Liu,

Schnell

2024

Preprint

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Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

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“…Antibodies labeled with small molecules are perhaps the most commonly used reagents in biology 1,2 . Antibodies labeled with fluorescent dyes and biotin are extensively used in imaging, immunoassays, flow cytometry, western blots, and immunoprecipitation among other applications [3][4][5][6] . Radiolabeled antibodies 3,7 find extensive use in imaging and therapy, antibodies labeled with cytotoxic drugs (ADCs) are offering new options for the treatment of cancers, and two ADCs have already been approved for therapeutic use 8…”

Section: Introductionmentioning

confidence: 99%

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Nath

Godat

Urh

2016

JoVE

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Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

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A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Cited by 5 publications

References 5 publications

Preclinical evaluation of the theranostic potential of 89Zr/177Lu-labeled anti-TROP-2 antibody in triple-negative breast cancer model

Preclinical evaluation of the theranostic potential of 89Zr/177Lu-labeled anti-TROP-2 antibody in triple-negative breast cancer model

Seeing Beyond the Blot: A Critical Look at Assumptions and Raw Data Interpretation in Western Blotting

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

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