3rd Annual 7 Steps of Protein Virtual Event

Protein sample evaluation is an important step in many protein workflows. The NanoDrop One microvolume spectrophotometer supports protein sample quantification with multiple preconfigured applications, including direct absorbance at A280, A205, or other wavelengths using the Thermo Scientific™ Pierce™ colorimetric assays (BCA, Rapid Gold BCA, Pierce 660, Bradford, etc.) and a proteins and labels application for quantitation of metalloproteins and fluorescently labeled proteins. We will review what you need to consider when choosing a protein quantification method for your sample. Is the sample one purified protein or a mixture of proteins? Does your peptide contain tyrosine and tryptophan residues? Does the sample buffer absorb in the UV spectrum? How can co-purified DNA affect your protein sample measurement? What is a good blank/control for your sample? Answers to these questions and other key considerations will be discussed.

Learning Objectives:

• Understand protein extinction coefficients—how to obtain the most accurate protein sample quantification using UV absorbance at 280 nm
• Learn how new Thermo Scientific™ Acclaro™ sample intelligence technology in the NanoDrop One spectrophotometer delivers information not only about the concentration but also the quality of your protein sample

Early detection of protein biomarkers is critical to the study of human diseases, yet current tools available for low-level protein quantitation do not address all the needs for protein and molecular biologists alike. A common concern is sample volume limitation due to difficulties in obtaining sufficient biological samples from various models in research studies. Additionally, cost and time-to-answer are always a factor when considering which assay platform to use. In many cases, end users must make trade-offs on which factors impact their work the least.

Invitrogen™ ProQuantum™ high-sensitivity immunoassays combine multiple benefits into a single platform so that users don’t need to make trade-offs. The platform works by combining the analyte specificity of high-affinity antibody–antigen binding with the signal detection and amplification of real-time PCR. The result is an assay that can measure low-expressing proteins (femtogram levels) while only consuming a fraction of sample volume (i.e.,1–2 microliters per sample) compared to other immunoassay methods. Best of all, it uses a streamlined workflow (2 hours) that is highly scalable and affordable on an open, widely used qPCR platform.

Learning Objectives:

• To understand the multiple benefits of ProQuantum high-sensitivity immunoassays and how the platform works
• To understand how the kits are designed for a simplified workflow amenable to high-throughput applications

Protein quantitation assays are essential for workflows that include protein extraction, labeling, and analysis. The simplest method for protein quantitation is measuring the absorbance of a protein sample at 280 nm (A280) and calculating the quantity using the Beer Lambert Law. However, this method is only applicable when the sample is a purified protein solution. Most cell biology experiments result in mixed or unpurified protein samples extracted from cell or tissue lysates. The Bradford assay (or Coomassie dye–based assay) has been a standard protein quantitation assay for unpurified proteins for some time, but a major drawback of this assay is its poor linearity. An improved assay, the Thermo Scientific™ Pierce™ BCA Protein Assay using bicinchoninic acid, is a chelator-based assay with exceptional linearity and low protein-to-protein variability that was quickly established as the “gold standard” for protein assays. One drawback with this assay, though, is the relatively long incubation time (30 minutes at 37˚C or 2 hours at room temperature). This poster presents the development of an improved BCA protein quantitation assay, the new Thermo Scientific™ Pierce™ Rapid Gold BCA Protein Assay, that addresses the need for a shorter incubation time (5 minutes at room temperature). Herein we present our evaluation of protein-to-protein variability, accuracy of “known” protein mixes, and closeness in protein estimation for this new assay as compared to the classic BCA assay. The results of this evaluation demonstrate that the new Pierce Rapid Gold BCA assay with the shorter incubation time provides the same accuracy as the original BCA assay.

Learning Objectives:

• A rapid way to quantitate protein using the same principle as the traditional BCA assay

To ensure the immune system’s balance between the recognition of non-self and the prevention of autoimmunity, the activity of immune cells needs to be strictly controlled. During the process of cancerogenesis, tumor cells try to resist immune responses and work to suppress the cycle of cancer immunity through multiple mechanisms involving regulatory proteins. These proteins—referred to as immune checkpoints—can affect immunoregulatory pathways by either boosting (co-stimulatory) or restricting (co-inhibitory) the immune response. For the majority of these checkpoint markers, soluble isoforms or shed variants that function as immune adjuvants or decoy receptors, have been identified. To date, cancer therapies that use antibodies targeted to the proteins within these immune checkpoint pathways have proven effective. It is also hypothesized that the measurement of the circulating concentration of the soluble forms of the immune checkpoint proteins could potentially correlate with the clinical efficacy of corresponding checkpoint modulator drugs.

To test this hypothesis, we developed two comprehensive multiplexing panels (using Luminex® xMAP® technology) that are specifically designed to detect soluble checkpoint molecules involved in T cell and NK cell regulation. The panels allow the simultaneous detection of immune stimulatory and inhibitory factors, allowing a more holistic picture of the molecular players in cancer immunity. Preliminary results using the Invitrogen™ Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex™ Panel 1 (Cat. No. EPX14A-15803-901) and the Invitrogen™ Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex™ Panel 2 (Cat. No. EPX140-15815-901) demonstrate that there are different expression patterns for each that are dependent on both disease state and patient treatment. More in-depth analysis of these soluble biomarkers will hopefully shed more light on the biology of the immune checkpoint pathways and provide a possible tool for monitoring response to therapeutic treatment.

Learning Objectives:

• Investigate the advantage of using soluble checkpoint molecules as potential markers for the response to chemotherapy
• Develop immunoassays for new immune regulating targets

Understanding the complex, yet coordinated protein interactions that sustain life, may help unlock the mysteries of disease mechanisms and thereby treatments. Protein identification and quantification have become extremely valuable in advancing our understanding and finding cures. As there are many methodologies available to scientists today, it can be challenging to choose which tools are the most effective to answer specific questions.

Based on the decades of market experience in the immunological research reagents market, Thermo Fisher Scientific addresses different customer needs by different technologies for detecting the same analytes and offers a vast choice of appropriate tools for the customer’s specific needs.

Learning Objectives:

• Identify time-saving technologies and tools for protein quantitation
• Understand the benefits of choosing from different immunoassay platforms and formats, all from the same provider