Protein Research Live

Successful scientific studies rely on reagent quality and reproducibility. As the market increasingly fills with reagent producers, there has been growing demand to apply additional levels of analysis to existing quality control assays. Biosensors have been used effectively in both academic and pharmaceutical development labs for many years to measure the binding properties of reagents. Recently, Bio-Techne began investigating product performance using SPR technology in a wide variety of assays. A novel surface has been developed which allows capture of any species antibody, thereby increasing efficiency and flexibility in assay design. Assays investigating natural and recombinant proteins and antibodies will be presented, as well as specific, customer-driven cases to support reagent performance.

G protein coupled receptors (GPCRs) are the targets of 30-40% of all approved drugs. However many approaches successfully applied to drug discovery for soluble protein targets have made a limited impact on GPCR’s due to challenges in obtaining pure, active, membrane free receptors for structural and biophysical investigation. Biophysical techniques in combination with improvements in protein engineering and handling have advanced to face the challenges of studying membrane bound GPCR’s with SPR in particular becoming increasingly utilized in the study of GPCR’s as the sensitivity of the detections systems has improved. Here we present the results from a collaboration with the Hopkins-Navratilova lab at the University of Dundee highlighting the importance of instrument sensitivity characterizing GPCR’s with the Biacore™ S200.

UNICORN™ software provides built-in knowledge for planning, controlling, and analyzing protein purification runs on ÄKTA™ chromatography systems.

With UNICORN 7, the evaluation module provides a simplified user interface optimized for most commonly used workflows such as:

• quick evaluation
• comparison of results
• work with peaks and fractions

Using the evaluation module, provides insights about protein purification runs and allow decisions to be based on rigid facts.

Join us for a presentation demonstrating how to gain quick insights to protein purification.

Size exclusion chromatography (SEC) is an extremely powerful technique to separate biomolecules based on differences in size under native conditions.

If you are using the SEC technique to purify or analyze proteins and other biomolecules, join this session.

Pia Liljedahl will introduce you to the new generation of agarose-based SEC columns from GE.

You will learn:

• how to choose the new generation resins and column formats according to your needs
• how these columns can impact the resolution and the run time of your experiment
• key applications in protein purification and analysis

The session will include a live Q&A session

CRISPR-Cas9 has increased the accessibility of genome engineering due to its ease of use and ability to cause double strand breaks (DSBs) at almost any locus of interest. DSBs are repaired in cells by two predominant pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). Endogenous repair of DSBs using the NHEJ pathway typically results in functional protein disruption (knockout) whereas the HDR pathway can be used to introduce exogenous genetic content (knockin). During this webinar we will focus on the utility of a synthetic dual RNA approach to apply CRISPR-Cas9 to HDR genomic engineering applications and will provide guidelines for improving CRISPR Cas9-assisted HDR. We will also discuss the use of short single-stranded DNA as a donor template for small insertions as well as plasmid DNA donor templates for large insertions. Lastly, we outline methods for characterization of HDR-generated cell lines for precise genomic engineering.

There is rapidly growing interest in using the CRISPR-Cas9 system for functional screening, both as a primary screening tool and as an orthogonal tool for RNAi hit validation. High throughput synthesis, combined with a proprietary algorithm that selects highly functional and specific guide RNAs, allows rapid generation of CRISPR RNA (crRNA) collections in arrayed formats. Arrayed crRNA screens offer the advantage of applying sophisticated assays, such as those requiring high-content microscopy, to investigate phenotypes that involve intracellular localization, morphological changes, or require time-lapse investigation. While pooled lentiviral sgRNA screens have demonstrated utility for loss-of-function studies, the types of assays used are typically limited to viability, sensitization, and some reporter assays. We will demonstrate the application of arrayed screening with synthetic crRNA libraries across multiple assay types, and present considerations for experimental success.

The use of CRISPR-Cas9 for gene editing has opened up many new avenues for scientific exploration around gene function. The rapid expansion of the field has led to a wide range of technology formats for use in both gene knockout and precision knockin genome engineering experiments. The seemingly ever-increasing toolset has also led to a widening knowledge gap for newcomers to the field to overcome in determining the proper reagents for performing experiments.

During this webinar we will discuss the basics of CRISPR-Cas9 gene editing and the key criteria and decision points in selecting reagents based on your desired application. We will review all types of guide RNA and Cas9 nuclease formats and discuss the advantages and disadvantages of each.

Furthermore, we will present techniques for optimizing experimental conditions to maximize success, including functional knockout and HDR for knockin. To provide context for the selection of different formats, we will present data showing direct comparison of Cas9 nuclease and guide RNA formats under different experimental protocols.

Development of biosimilars for marketed bio-therapeutics is expected to increase patient access and affordability of these therapeutics within the healthcare system.  One of the key aspects of establishing biosimilarity at the analytical level is a comparative assessment of binding properties of the proposed biosimilar candidate and reference product. With a high level of sensitivity, and ability to measure kinetic parameters, Surface Plasmon Resonance (SPR) is an important analytical tool for characterizing binding properties of biological products. This presentation outlines strategies for biosimilar characterization utilizing various SPR methods. Challenges associated with day to day variability and statistical evaluation of data are summarized. Assay qualification efforts to make SPR assays more robust and repeatable, as well as solutions for better use of SPR as a part of biosimilar characterization are discussed.

Biosensors are now a well-established technology in the study of small molecules.  This talk will focus on several aspects of this type of work that biosensor users can be expected to explain.  These are: A) Describing Mass Transport: Mass transport is a common phenomenon in biosensor data that can affect the uniqueness of solutions derived from fits.  The origins of mass transport will be described as well as how mass transport has been modeled quantitatively to help explain the lack of uniqueness in solutions.   How mass transport manifests itself with respect to the shape of sensorgrams will also be described. B)  Reproducibility of Biosensor Data: Biosensors are commonly used to derive rate and equilibrium constants for molecular interactions.  Many of these values appear in publications and technical reports.  A commonly asked question about this technology relates to the magnitude of experimental errors that can typically be expected.  Some case examples will be discussed to help orient the audience as to the reliability of values of cited. C) Problematic Data:   Several case examples of actual problematic data that has been collected will be presented.  The dilemma that some of these data sets can pose in their interpretation will be discussed.

This is an introduction to the biological mechanism of RNA interference (RNAi) and experimental application of short, interfering RNA (siRNA). The key steps of the endogenous RNAi pathway will be explained to provide context for the mechanism of targeted gene knockdown with siRNA. The critical factors that affect siRNA function and specificity (off-targeting), such as siRNA design, chemical modifications and concentration, will be covered in detail. The practical application of siRNA will be covered through discussion of best-practice for experimental design. This will include proper use of positive and negative controls, optimization of transfection, and siRNA concentration. Measurement of gene knockdown and appropriate interpretation of experimental outcomes will round out the discussion. In this webinar, attendees will learn:

• Key steps of the endogenous RNAi pathway
• siRNA mechanism of action
• Best practices for siRNA experimental design
• Appropriate interpretation of experimental outcomes

Development of robust purification processes requires significant time and resources. In addition, steadily increasing demands from regulatory authorities for better understanding and control of manufacturing processes put even more pressure on development work. High-throughput process development (HTPD) shortens development time at the same time as it increases the amount of information available during early process development.

Here we will present a case study using high throughput process development tools optimizing process conditions for a mAb polishing step using a multimodal chromatography resin (Capto MMC ImpRes)

Significant improvements in a chromatography workflow can be achieved with small changes and proactive choices.

Before starting protein purification, it is important to define your requirements and objectives. These may include outlining the required purity, activity and the quantity of the target you want to achieve, ensuring process steps and handling are kept to a minimum and all critical impurities are removed.

This educational session is aimed to give guidance to protein purification protocol development for laboratory scale, and is designed for researchers working with either tagged or native proteins.

A key component of this Webinar is the introduction of a proven protein purification strategy, called CiPP (Capture, Intermediate purification, Polishing). It explains how to best combine the main separation techniques in order to obtain a powerful purification protocol delivering the pure and active protein you need for your work.

The aim with this session is to give a brief overview of various parameters that you need to consider when deciding your strategy how to obtain quantitative data.

The CRISPR-Cas9 system is being widely used for genome engineering in many different biological applications. It was originally adapted from the bacterial Type II CRISPR system and uses a Cas9 endonuclease guided by RNA to introduce double-strand DNA breaks at specific locations in the genome. The Dharmacon Edit-R CRISPR-Cas9 Gene Engineering platform includes the three components required for gene editing in mammalian cells: (1) a plasmid expressing a Cas9 nuclease, (2) a chemically synthesized tracrRNA, and (3) a synthetic crRNA designed to the target site of interest. In this presentation, the complete workflow of the Edit-R platform is demonstrated to knock out the PPIB gene at the protein level in HEK293T cells. Data from the analysis of 42 edited clones are presented.
  
Attendees will learn:

• CRISPR-Cas9 background and crRNA design considerations
• Transfection optimization for maximal editing efficiency
• Enrichment of cell populations with gene editing events by FACS
• Clonal selection
• Detailed analysis of editing events

SPR technology is being improved steadily and today is an essential component in the biopharmaceutical industry. As SPR based assays extract valuable and reliable information at high accuracy and precision they also provide huge advantages and opportunities for basic and applied research. Progress in synthetic biology and scalable transient gene expression systems is rapidly expanding not only the demand but also increasing the repertoire and quality of reagents for molecular recognition studies. We use SPR assays for a huge variety of tasks within a multi-user facility and are particularly interested in using plant-based systems for the discovery, development and production of biopharmaceuticals. The practical uses and applications to plant-produced antibodies and malaria vaccine development will be illustrated.

Focusing on protein cocktails, questions addressing their identity, composition, and potency will be discussed. SPR based assays are not only considered to be instrumental to advance such products into the clinic but will play an equally important role for studying their behavior and efficacy in patients.

Significant improvements in a protein purification workflow can be achieved with small changes and proactive choices.

This applies for reaching purification objectives: getting the right amount and quality of target protein from smoothly operating chromatography system, columns, and media (resins).

During this session, Dr Danielsson will share hints and tips about how to

• improve the yield and purity of the target protein
• make sure the chromatography system runs smoothly leading to increased lab efficiency and better results.

First established in the 1880’s, cell culture is the process by which cells are grown in a laboratory under controlled environment. The culturing of different kinds of cells supports scientific, technological and therapeutic developments in human and animal health. Technology has allowed the production of a diverse number of biologic compounds such as monoclonal antibodies, vaccines, and cytokines. Given their importance, cell culture strategies have been developed that address the environmental conditions and nutritional demands for optimal metabolism and proliferation of cell lines for different applications.  
 
We will discuss vital elements to successful cell culture including the choice of medium that provides the necessary nutrients to the cells. Media range from classical formulations to those designed to promote cell growth that are of animal-free origin. Media formulations also include those that can promote cell growth in the absence of serum supplementation and those that are chemically defined. GE Healthcare provides such media for many of the cell culture dependent biotechnology applications.
 
While media is an important component of successful cell culture, reliable results and optimal production can only be achieved while maintaining excellent control over the process of cell growth, which is susceptible to contamination by various sources. We review critical aspects of prevention with simple techniques and good laboratory practice.