Biophysical characterization of protein reagents for batch-to-batch reproducibility

The quality of protein reagents is critical to ensuring reproducibility and consistency for research. In this webinar, Deborah Moore-Lai briefly introduces a new protein product line and discusses the approach taken at Abcam to ensure batch-to-batch consistency of these proteins.

This reproducibility is validated through extensive biophysical quality control, including reversed-phase HPLC, intact mass analysis, and thermal ramp stability analysis. Using thermal stability, Abcam is able to further demonstrate batch-to-batch reproducibility using a correlation coefficient, which is generated by comparing thermal stability between individual protein batches. The full biophysical data analysis is provided for users to demonstrate Abcam’s commitment to quality and consistency, and to contribute towards successful research outcomes for its customers.

Read in for highlights from the live Q&A session or register to watch the webinar at a time that suits you.

What proteins do you offer beyond cytokines and growth factors?

DML: We’re starting to look at a variety of intracellular proteins involved in immune responses and transcription factors. The portfolio right now is focused on cytokines and growth factors, but over the next year or two, we are broadening that portfolio to more proteins, such as transcription factors, and other key regulators of the immune system.

I've heard about large value orders (LVOs). What does this mean? And how do I inquire about one?

DML: An LVO is just something that is not commercially available on the website. Depending on the size that you need, we'll produce that for you. If you need 1 milligram, 3 milligrams, 10 milligrams of protein, all of those would constitute an LVO. We are happy to talk with you and work with you on that, including if you needed a different formulation.

How do I select the most appropriate protein expression system?

DML: It depends on your research needs and the protein itself. For example, if the protein is intracellular and does not have disulfide bonds or post-translational modifications, a prokaryotic bacterial system will suit your needs. If, on the other hand, your protein is complex and it is secreted, and it has disulfide bonds that you would prefer that the protein is expressed with, then mammalian is likely a better option for you.

Insects can perform post-translational modifications and folding. The yield from an insect system can be a little bit lower than mammalian but we would try E. coli if the protein meets those requirements, if not, we'll go mammalian and insect in the same trajectory.

How do you remove endotoxins in your sample?

DML: My recommendation is don't have endotoxins there in the first place. The lab in Waltham is exclusively mammalian, and so it's very, very clean. The instruments are sanitized weekly with a really deep sodium hydroxide clean to ensure that there's nothing growing in them over the weekend. We do have removal methods if there is endotoxin in the sample, but you take a hit on your protein yield. My recommendation is just to use ultra-pure reagents.

We have very high-quality water in the lab for proper preparation. Often, what you see with endotoxin is it seems to just bind to some proteins irreversibly. So once it's bound, there's nothing you can really do. The best practice is to just be very hygienic, upstream before purification and then during.

How do you pick which cell-based assay to use for your proteins?

DML: What we do is look at the literature and look for the gold standard assay, and then we proceed accordingly. In terms of difficulty, proliferation assays are the most straightforward for the lab to run and that would be ideal. Sometimes it's chemokines, and obviously, we need to do a chemotaxis assay. We find those are some of the hardest and most difficult assays to run.

We see what our competitors are using for whatever that gold standard assay is, and then we proceed accordingly. And we will also often include a calibrator protein just as a positive control to understand if our protein is behaving properly or if it's something about the assay itself, and the setup of the assay that we need to redesign.

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