Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Wanandy, Troy; Handley, Simon A.; Mulcahy, Emily; Wiese, Michael

doi:10.1016/j.toxicon.2024.107685

Cited by 1 publication

(1 citation statement)

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“…This is particularly important when new trainees are assigned to run protein assays, increasing the risk of pipettor or dilution errors that can compromise results. While a number of publications have compared different quantitation methods, the importance of preparation and storage methods for the assay standards has generally not been addressed 1-4 . Some publications and online guides indicate that storage conditions and freeze-thaw cycles of proteins result in degradation of protein due to denaturing through temperature-induced structural changes, protein aggregation, and changes in concentration during freeze thaw events; however, these reports typically look at complex mixtures of proteins isolated from cells or tissue and not a purified single protein such as those used as standards 2 .…”

Section: Introductionmentioning

confidence: 99%

Freezing Diluted Bovine Serum Albumin Standards Does Not Significantly Affect Standard Curves

Sheets,

Rajaboina,

Bromberg

et al. 2024

Preprint

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Total protein isolation followed by quantitation is a common protocol in many laboratories. Quantitation is often done using a colorimetric assay such as the bicinchoninic acid (BCA) assay in which a change in the color of the BCA reagent is related to protein concentration. Extracted protein samples are compared to a standard curve made with dilutions of a protein standard such as bovine serum albumin (BSA) to determine their concentrations. A series of experiments was designed to determine the most reproducible and accurate method for quantifying protein concentrations of samples in an experimental series over time. The effect of freezing on diluted standards was investigated. Standards were frozen at −20°C or −80°C and serially thawed and refrozen up to three times prior to their use in a BCA assay. Thawing and refreezing the standards had no significant effect on protein concentration and the resulting standard curves. Inter-person and intra-person variability in the preparation of standards was also investigated. Protein concentration differences due to inter-person and intra-person variability were greater than protein concentration variability resulting from freezing and thawing, regardless of the freezing temperature. The most reproducible and accurate method for determining the protein concentration of extracted samples in an experimental series over time is diluting a large batch of BSA standards and freezing them at either −20°C or −80°C. Reproducibility was maintained with up to three freeze-thaws.HighlightsFreezing diluted BSA standards at either −20°C or −80°C and thawing and refreezing them up to three times does not significantly alter their protein concentrations.Inter-person variability in standard curves is greater than intra-person variability, and controlling for developer decreases variability in most cases.There is more consistency in standard curves when a single batch of diluted standards is aliquoted and frozen at either −20°C or −80°C and thawed up to three times than when the same investigator or different investigators make fresh standards before each assay.

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Section: Introductionmentioning

confidence: 99%