The Orbion Blog
Protein Science, in Practice
Field-tested deep-dives on structural biology, protein engineering, expression, and the AI behind it all — written for the people at the bench
TEV, HRV 3C, Thrombin, or SUMO Protease: Choosing a Tag-Cleavage Strategy
The fusion expressed, the affinity step worked, and then tag removal turned one clean band into three. Protease choice is often made from a vector map before…
Orbion Is Now ISO 27001 Certified
Before you paste a sequence into any tool, you ask the quiet question: where does this go, and who can see it? For a protein engineer that is not paranoia.…
Anion or Cation Exchange? A Practical Decision Tree for Protein Purification
Your target has a predicted pI of 6.4. Should it go on an anion exchanger at pH 7.5, a cation exchanger at pH 5.5, or neither? The textbook answer—work above…
Nickel vs Cobalt Resin for His-Tagged Proteins: Purity, Yield, and When to Switch
Nickel resin gives you 18 mg of target plus a forest of host-cell proteins. Cobalt resin gives you 7 mg and a cleaner lane. Which one is better? The answer…
Why DNA and RNA Co-Purify With Your Protein: Fixing High A260/A280 and Viscous Lysates
Your lysate pulls like syrup, the affinity column pressure climbs, and the “pure” elution has an A260/A280 ratio close to 1.0. The gel may show one dominant…
Working With Intrinsically Disordered Proteins: Expression, Purification, and Why AlphaFold Fails on Them
Your 18 kDa intrinsically disordered protein runs near 30 kDa on SDS-PAGE, elutes from size-exclusion chromatography where a much larger globular protein…
Protein Language Models vs MSA-Based Predictors: Which to Trust for Your Target
AlphaFold gives your orphan protein a soft, low-confidence fold after searching a shallow alignment. ESMFold returns a compact model in seconds with pLDDT…
Reconstituting a Membrane Protein into Nanodiscs: A Step-by-Step Troubleshooting Guide
You mix purified receptor, MSP1D1, and POPC at the ratio that worked in a paper. After overnight detergent removal, the tube is clear. The SEC trace is not:…
Solubility Tags for Difficult and Membrane Proteins: When MBP, SUMO, or Fh8 Actually Help
Your His-tagged construct is almost entirely in the pellet, so you replace His6 with MBP. The next expression test looks transformed: the fusion is abundant…
AlphaFold Says High Confidence, but Your Protein Misbehaves: Reconciling pLDDT with Bench Reality
Your AlphaFold model is blue from end to end. Median pLDDT is 92, the core looks compact, and the active-site residues sit exactly where the literature says…
Detergent Selection for Membrane Protein Extraction: A Decision Tree from DDM to LMNG
Your membrane prep contains the target. One percent DDM moves most of it into the supernatant. The yield looks excellent—until the sample reaches immobilized…
Rigid or Flexible? How to Choose the Linker Between Your Tag and Your Protein
Your fusion expresses beautifully. The gel shows a crisp band at the right size, the His-tag pulls it down cleanly, and yield is high. Then you run the…
How Many Homologs Does AlphaFold Need? MSA Depth, Orphan Proteins, and Single-Sequence Prediction
Your model came back at pLDDT 45 across almost the entire chain. No confident core, no floating disordered tail you can point to, just a uniform wash of…
GroEL Is in Your Prep: Why Chaperones Co-Purify With Your His-Tagged Protein
Your His-tagged protein came off the Ni-NTA looking clean, mostly. There is your band at the expected mass, and then there is that other one: a fat, stubborn…
N-Terminal or C-Terminal? Where to Put Your Tag (and Why It Changes Everything)
You designed two constructs of the same protein with the same His10 tag. The only difference: one carries the tag at the N-terminus, the other at the…
AlphaFold Gives You One Structure. Your Protein Has Several.
You typed in a sequence, waited a few minutes, and got back a single, beautiful model with a 90-plus pLDDT core. It looks definitive. It looks like the…
Why Won't My Protein Concentrate? Precipitation at High Concentration, Explained
Purification went flawlessly. The SEC trace is a single symmetric peak, the A280 says you have 8 mg spread across 12 mL, and all you need is 5 mg/mL for the…
Which PTMs Actually Survive in Your Expression Host? How to Read a PTM Prediction Before You Pick E. coli or HEK293
You ran your sequence through a PTM predictor and got back a clean list: two N-glycosylation sequons, a cluster of phosphosites in a cytoplasmic loop, three…
Your Disulfide-Rich Protein Won't Fold in E. coli: The Redox, Strain, and Periplasm Decision Guide
You subcloned your antibody fragment — or your growth factor, or your protease inhibitor — into a clean pET vector, transformed BL21(DE3), induced, and ran a…
ΔTm or ΔΔG? Which Stability Metric to Trust When You're Ranking Mutations
You ran a stability predictor on 40 candidate mutations, and now every row has two numbers. One mutation shows a predicted ΔTm of +6.2°C but a ΔΔG of only…
Your Protein Expressed as Inclusion Bodies — Refold, Redesign, or Re-Host? A Decision Guide
Your SDS-PAGE says the protein expressed beautifully — a fat band at exactly the right molecular weight. Your supernatant says otherwise: it's clear, and…