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Protein Characterization

Analytical characterization ensures the identity, purity, structural and conformational integrity, and function of the protein. We perform a number of routine protein analyses throughout all project stages. If needed, we can submit your samples for additional methods of characterization. We also offer specialized protein characterization services for purified proteins.



ANALYSIS OF RAW, INTERMEDIATE AND FINAL PROTEIN PRODUCTS

  • electrophoresis (SDS-PAGE, native-PAGE, IEF-PAGE, urea-PAGE)
  • Western blot/dot blot
  • enzyme activity assay by light absorbance or fluorescence
  • ELISA (direct or sandwich)-ELISA Development
  • protein assay (A280, BCA or equivalent)
  • antibody isotyping
  • endotoxin measurement -Endotoxin Assay Quote Request
  • contaminating DNA assay
  • UV-Vis absorption spectrum

PREPARATION AND SUBMISSION OF PROTEIN SAMPLES FOR

  • amino acid analysis
  • N-terminal analysis
  • mass-spectrometry identification and analysis
  • extinction coefficient determination

PROTEIN CHARACTERIZATION

  • analytical size exclusion chromatography (native MW estimate, aggregation analysis)
  • analytical ion-exchange chromatography (oxidation)
  • purity analysis by densitometry
  • UV-Vis light absorption spectroscopy
  • analysis of oxidation, degradation and aggregation products
  • protein deglycosylation analysis
  • binding interactions by co-immunoprecipitation, spectroscopy or chromatography

Useful Tools for a Protein Characterization Project

Protein Concentration Calculator
Antibody Dilution Calculator
Molarity to mg/ml Calculator


Selected Latest Developments in Protein Characterization


Multi-domain unfolding of the Fab fragment of a humanized anti-cocaine mAb characterized by non-reducing SDS-PAGE

Kirley & Norman, Biochem Biophys Res Commun, 2020 Dec 10;533(3):580-585.
Abstract
Monoclonal antibodies and their fragments are widely used for research and therapy. Fab fragments are useful since they retain antigen binding specificity, but being smaller proteins, are better able to penetrate biological compartments and tumors, and do not induce Fc-dependent immunological system activation. Our laboratory developed an anti-cocaine mAb (named h2E2) for the treatment of cocaine use disorders, which is currently in advanced pre-clinical development. We are also interested in the Fab fragment of this mAb for potential therapy of acute cocaine overdose. Previously, we showed that this mAb and its F(ab')2 and Fab fragments undergo discrete domain unfolding, as detected by non-reducing SDS-PAGE, and that ligand-induced protein thermal stabilization can be quantitated utilizing differential scanning fluorimetry in the absence of SDS. Here, we demonstrate that multiple Fab protein gel bands observed using non-reducing SDS-PAGE in the presence and absence of cocaine and its metabolites can be explained and interpreted based on the differential stabilization of two unfolding domains in the Fab fragment. The variable domain is stabilized by ligands against SDS unfolding, while the constant domain is not. This data and its interpretation are also supported by differential scanning fluorimetry data for the Fab fragment in SDS. It is likely that these non-reducing SDS-PAGE results and the gel band domain unfolding model will be applicable to other small molecule binding antibodies. Thus, non-reducing SDS-PAGE is a widely available and simple method for assessing domain stability and multi-step unfolding of Fab fragments.
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BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow.

Ayako Takemori et al., J Proteome Res, 2020 Dec 24.
Abstract
The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.
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A systematic approach to quantitative Western blot analysis

Lakshmi Pillai-Kastoori et al., Anal Biochem, 2020 Mar 15;593:113608.
Abstract
Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize sources of error and variability throughout the Western blot process.
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