Protein Characterization

Protein characterization is crucial in life sciences research, covering detailed aspects like protein structure, function, and purity. It's key to advancing drug development and producing biological products effectively.

Creative BioMart utilizes advanced platforms-mass spectrometry, SPR, and Cryo-EM-along with our vast experience to offer top-notch protein characterization services. We aim to support our clients in meeting diverse research and application goals, ensuring they receive insightful and dependable results.

Overview of Protein Characterization

Why Protein Characterization Matters?

Protein characterization is vital in contemporary biomedical and life science research. It involves examining a protein's structure, role, purity, and stability. This analysis is fundamental in drug development, biological product manufacturing, vaccine creation, and antibody refinement. Through in-depth protein analysis, researchers can uncover protein functions, potential activities, and interactions with other molecules. This insight helps in drug discovery and developing biotech products. Additionally, protein characterization is widely applied in disease diagnosis and precision medicine, helping identify new biomarkers and develop personalized treatments.

Key Technologies & Methods

Protein characterization taps into a variety of cutting-edge techniques to help scientists understand proteins from different angles. Here’s a look at some key methods:

• Mass Spectrometry (MS)

  Think of MS as a magnifying glass for proteins-it’s essential for figuring out the nitty-gritty details like molecular weight, sequences, and any modifications. It’s a big hit in proteomics.

• Surface Plasmon Resonance (SPR)

  SPR lets researchers check out how proteins mix with other molecules, such as ligands and antibodies, providing real-time insights without needing any extra labels.

• Cryo-Electron Microscopy (Cryo-EM)

  Cryo-EM dives into high-res 3D views of proteins, giving fresh perspective into their structures and helping scientists understand how they work.

• Bio-Layer Interferometry (BLI)

  BLI is all about pinpointing how tightly proteins and other molecules bond. It’s especially handy for looking at antibodies, vaccines, and protein-small molecule interactions.

• Yeast Two-Hybrid System

  This tool helps researchers find and study interactions, particularly useful for diving into cell signaling and disease-related processes.

What We Offer?

Our Protein Characterization Services

Preparation and submission of protein samples for

• Protein identity, purity and impurities
• Charge isoforms
• Oxidation
• Aggregation analysis
• Amino acid analysis
• Peptide mapping
• Glycan analysis and profiling

Aspects	Platform and Analytical Techniques
Protein Identity, Purity and Impurities	SDS electrophoresis Analytical Size-Exclusion Chromatography Chip based Protein Electrophoresis Assays Capillary electrophoresis UV detection HPLC
Charge Isoform	Gel Isoelectric Focusing Capillary Isoelectric Focusing Whole-Column Isoelectric Focusing OFFGEL electrophoresis Ion Exchange Column-HPLC
Aggregation Analysis	SDS PAGE Chip-based Protein Electrophoresis Size Exclusion HPLC Light Scattering
Oxidation	HPLC-LC/UV Reversed-Phase LC/MS Enzymatic digestion + LC/MS
Amino Acid Analysis	OPA derivatization + RP-LC/UV FMOC derivatization + RP-LC/UV Derivatization + RP-LC/MS
Peptide Mapping	RP-LC/UV RP-LC/MS Capillary electrophoresis detection by DAD (diode array detection) or MS
Glycan Profiling	HPLC/fluorescence detection CE/fluorescence MALDI-TOF/MS LC/MS HPLC/pulsed amperometry detector

Service Workflow

Service Procedure	Technical Methods
Requirements Analysis	Experiment Consulting and Design Service
Protein Expression and Purification	Protein Expression and Purification Services	Bacterial expression Systems ( E. coli / Bacillus )
		Yeast expression Systems ( P pastoris / S cerevisiae )
		Baculovirus-Insect Cell expression Systems (SF9, SF21, S2, T.ni / HI-5)
		Mammalian expression Systems (CHO / HEK293)
	Protein Engineering Services	Directed evolution
		Rational design
		Library construction
	Membrane Proteins Expression and Purification	Nanodisc Technology Platform
		Liposome Technology Platform
		Virus-Like Particles (VLPs) Technology Platform
		FoldEZ™ Platform
		LiPi™ Technology Platform
	Protein Extraction Services	Organelle protein extraction
		Nuclear protein extraction
		Membrane protein extraction
		Cytoplasmic protein extraction
	Endotoxin Removal Service
Protein Function and Modification Analysis	Protein Modification	Glycosylation , phosphorylation, methylation, ubiquitinylation and acetylation. Redox modification of proteins, protein engineering and protein labeling .
	Protein Function	Protein Sequence Analysis and Function Prediction
	Protein Interaction	Yeast two-hybrid
		Membrane-based yeast two-hybrid
		Mammalian two-hybrid
		Surface plasmon resonance (SPR)
		Fluorescence resonance energy transfer (FRET)
		Protein array
		CLIP-Seq technology
	Cell-Free Based Protein Analytical
Protein Quantitative Analysis	Active Concentration Analysis
	Standard Concentration Measurement
	iTRAQ Multiplex Protein Quantitation
Protein Characterization
Biomarker Discovery

Instrument Platform

At Creative BioMart, we're all set with top-notch tools to offer a full suite of protein characterization services. We've got things like mass spectrometry (MS), surface plasmon resonance (SPR), cryo-electron microscopy (Cryo-EM), and bio-layer interferometry (BLI). Plus, we've got the yeast two-hybrid system, high-throughput screening setups, nuclear magnetic resonance (NMR), X-ray crystallography, differential scanning calorimetry (DSC), and dynamic light scattering (DLS). With these tools, we can dive deep into studying protein structures, functions, purity, stability, interactions, and even post-translational tweaks.

Case Study

Fig. 1. Top 4 most prominent proteins identified in 12 bands from day-one eggs.

Case 1: Analyzing Tick Egg Protein Changes Using SDS-PAGE and LC-MS/MS (Tang Q.; et al. Animals (Basel) . 2024)

Tick eggs have proteins vital for their development. Understanding these proteins is key to studying tick embryogenesis and finding new treatment targets. This study looked at eggs at four stages (0, 7, 14, and 21 days of incubation) using SDS-PAGE to analyze protein profiles. On day one, proteins were re-analyzed with LC-MS/MS. Forty protein changes during development were explored using LC-PRM/MS. In day-1 eggs, 108 transcripts were found and grouped into categories like transporters, enzymes, immune proteins, and more. Vitellogenin and vitellin-A dominated nine protein bands, with others being cathepsins and Kunitz proteins. The analysis showed an increase in 28 transcripts, including enzymes, antimicrobial peptides, and heat shock proteins, while five transcripts like Kunitz proteins and proteases decreased. This research offers an overview of egg proteins and their changes during development, aiding in understanding protein roles and pinpointing study targets.

Fig. 2. Non-reducing SDS-PAGE analysis of rFVIIIFc validation batch (RECD19189-11-011) used for the determination of purity and identity.

Case 2: Producing and Ensuring the Purity of Long-Lasting rFVIIIFc for Hemophilia A Treatment (McCue J.; et al. Biologicals . 2015)

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-lasting clotting factor used for hemophilia A treatment. This piece covers how rFVIIIFc is made and checks for quality, impurity removal, and virus clearance. The process is designed to be scalable, using multiple purification and virus-clearing steps, like a unique affinity adsorbent and a 15 nm virus filter. rFVIIIFc is produced from human embryonic kidney (HEK) 293H cells. Tests confirmed its identity, purity, activity, and safety. The process reliably removes impurities and viruses, achieving over 8-15 log ¹⁰ clearance for four test viruses. Non-human glycans, like terminal galactose and N-glycolylneuraminic acid, weren't found in rFVIIIFc. Overall, the process delivers a pure, virus-free product with the capability to meet supply demands.

Fig. 3. Methylation of H2B (23-37) peptide is not altered by the addition of Fhod3 N-terminus.

Case 3: Cross-Talk Between Phosphorylation and Methylation in Fhod3 Protein Regulation (Lowe TL.; et al. J Biol Chem. 2024)

Protein post-translational modifications (PTMs) adjust biological processes by altering amino acid properties like size, charge, and bonding. Typical PTMs include phosphorylation, methylation, acetylation, and ubiquitylation. While studying a single modification often takes center stage, other changes can add complexity. For example, arginine methyltransferases and serine/threonine kinases might target similar motifs, leading to enzyme cross-talk. This study discovered that formin homology proteins (Fhods) have a motif specific to PRMT7, which methylates arginines R1588/R1590 in Fhod3. S1589 and S1595 can be phosphorylated by Rho/ROCK1 kinase. Notably, phosphorylation at S1589 blocks PRMT7 methylation at R1588/R1590, but methylation has little effect on ROCK1. Also, the N-terminal DID domain can inhibit DAD domain methylation.

Fig. 4. Multiple sequence alignments of A. catechu and 24 subgroups A. thaliana . (partial)

Case 4: Exploring bHLH Transcription Factors in Areca Catechu (Ali A.; et al. Int J Mol Sci. 2024)

The basic helix-loop-helix ( bHLH ) transcription factor family ranks as the second-largest among eukaryotes, known for its diversity in plants. Yet, Areca catechu ’s bHLH genes remain unexplored. This study identified 76 AcbHLH genes, detailing their physical and chemical traits. Researchers examined their evolutionary connections with Arabidopsis thaliana ( AtbHLH ) and uncovered shared amino acid motifs like S, R, K, and P. The promoter analysis revealed 76 growth-related elements. Gene duplications showed four tandem and twenty-three segmental duplications, with AcbHLH63 displaying strong links with bHLH genes from multiple plants, including A. thaliana. Under drought stress, genes such as AcbHLH22 and AcbHLH39 are more active in leaves, with all nine genes more active in roots. Salt stress triggered similar responses in these genes across different times.

Fig. 5. Peptide color mapping along the sequence of the major parent proteins of HM and IF.

Case 5: Peptide Mapping and Digestion Comparison of Infant Formula and Human Milk (Charton E.; et al. Food Res Int. 2024)

Infant formula (IF) aims to replicate human milk (HM) but still needs improvements in digestion. This study used mini-piglets and the DIDGI® lab model to see how IF and HM break down. Researchers tested fresh HM and standard bovine IF. Young piglets were fed HM or IF, and their digestive process was studied. In vitro , digestion mimicked a four-week-old infant. Analyzing structure and protein breakdown, both methods showed HM and IF digest differently. HM’s protein digestion was slower than IF’s. About 40-50% of bioactive peptides in live tests correlated with in vitro results, highlighting distinct digestive paths for HM and IF.

Why Choose Us?

Professional Know-How and Technical Edge
At Creative BioMart , we've been working in protein characterization for years. Our expert team and cutting-edge tech mean we can deliver top-notch protein analysis for clients all over the world.

State-of-the-Art Technology
We've got the latest tools like mass spectrometry, SPR, and Cryo-EM to dig deep into protein structures, functions, and interactions-ensuring you get high-quality data.

Customized Solutions
We tailor our services to fit what you need, offering support from the beginning with sample prep right through to data analysis, making sure your project’s specific needs are met.

Quality Assurance and Reliability
By following global standards, we promise that our services are accurate and repeatable, giving you data you can trust for your research.

Broad Industry Use
Our services are a big hit in drug and vaccine development, as well as antibody research, helping speed up scientific breakthroughs and foster innovation.

FAQs

• Q: Which proteins do Creative BioMart's characterization services include?

  A: We handle different types of proteins, including monoclonal and polyclonal antibodies, recombinant proteins, natural proteins and fusion proteins, and are adept at handling a wide range of structures and complexities.

• Q: Can I choose just a specific type of analysis service?

  A: Yes, we offer custom services where you can pick specific characterization options, like peptide mapping or glycosylation analysis, or go for a full comprehensive analysis.

• Q: What are the sample requirements when providing them?

  A: At the start of the project, we'll provide detailed sample requirements, including purity, concentration, and volume. You can also talk directly with our technical team.

• Q:What reports and data will I receive after the service is completed?

  A: You'll get a full report that includes technical details, result analysis, mapping data, and a summary, all ready for your further research or applications.