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More from @DeryaTR_

Derya Unutmaz, MD

@DeryaTR_

Sep 3

I am now sharing an extremely detaild new method invented by the OpenAI GPT-5 Pro model for one of biology’s oldest and most foundational techniques: Western blotting. I had the honor of learning it in 1989 in Switzerland from two of its original inventors, Drs. Harry Towbin and Theo Staehelin.

This AI-generated scientific method aligns with what @kevinweil announced today about starting OpenAI for Science, an AI-powered platform aimed at accelerating scientific discovery. It also supports what I have long argued: models like GPT-5 Thinking and Pro can accelerate the scientific process by orders of magnitude; this is another example!

It is important for non-scientists to realize that technology drives science. Without novel scientific techniques and methods, we cannot sustain accelerated discovery. This is especially true in biology, which is extraordinarily complex, with trillions of parts and dynamically interacting processes. Without AI we would have little hope of understanding all this complexity and solving biology, curing all diseases, or reversing aging in the coming decades, or even centuries.

This is why I've always been so exicted about AI and went all in several years ago after ChatGPT release, when it became clear to me the path to AGI has been opened. It is now time to recognize that this is not a hypothetical future any more; it is already here and getting better by the month, week, and day!

I should also point out that further development and proof-of-concept experimentations could turn this particular method into a patentable invention. However, I strongly believe that in the age of AI, such inventions should not be patented and should belong to all of humanity. In fact, our patent and intellectual property laws need to be substantially modified, or even replaced, to align with the reality that soon most inventions may be generated by AI, with human contribution increasingly focused on application and value creation. I digress; that is a deeper topic for another time :)

Before I share the method developed by GPT-5 Pro in the following thread, I would like to provide some historical background and explain the importance of the Western blotting method, which every biologist knows or should know. Here I am sharing a summary of GPT-5’s explanation; of course, anyone can check with Grok or other models for more detail:

What is Western Blotting?
Think of a cell extract as a soup full of different proteins. Western blotting does four simple things:
Sorts by size. An electric field pulls proteins through a jelly-like slab. Small ones run farther than big ones.
Copies the pattern to paper. The separated proteins are moved onto a thin membrane that holds them in place.
Finds your protein with a matching “key.” An antibody that sticks only to your protein is added, then a second antibody with a built-in label makes the spot light up or change color. The position tells you the protein’s size, and the brightness tells you roughly how much is there.

Who invented it, and when?
In 1979, two groups independently created the core method of protein “blotting” to a membrane after gel electrophoresis: Harry Towbin, Theophil Staehelin, and Julian Gordon in Basel, and Jaime Renart, Jakob Reiser, and George Stark at Stanford. Two years later, in 1981, W. Neal Burnette popularized the name “western blot.”

Why is it a pillar of biological methods?
Historically and practically it is one of the core lab methods for protein analysis. The reason is its specificity with size information. It tells you not just that a protein is present, but also its approximate size, which helps confirm identity, isoforms, and post-translational modifications. Indeed, Western Blotting remains one of the most commonly used protein assays across research fields and has been central to countless studies and workflows. Labs use it to verify antibody targets, confirm expression or knockdowns, and check pathway activation and use it in diagnostics (for example early HIV testing was dependent on this).

I will share the detailed method in the next thread. It is important to point out that this is a very sophisticated method, that took several prompts to validate. However, it can continue to be improved and optimized further and of course significant lab effort will be needed to develop it and troubleshoot it. This is a very detailed blueprint, which in itself is extraordinarily remarkable! In the third thread below I provide some critiques (also from GPT-5 Pro) and how to further validate and further improve it.

Because this is very technical it may be quite difficult for those not familiar with these type of methods to follow, so here is a scientific and layperson’s summary of the gist:

DSI-Seq (novel western blotting replacement)

Scientific summary of DSI-Seq: Digital Size-Indexed ImmunoSequencing transforms the Western blot from an artisanal, low-plex picture into a standardized, high-plex, quantitative assay that preserves size-resolved proteoforms. By coupling rapid microchip SDS separation and inline fractionation to DNA-barcoded immunoassays with UMI-based counting, DSI-Seq delivers isoform-aware pathway readouts from tiny inputs with built-in controls and shareable digital outputs. This capability enables rigorous kinetic mapping of signaling, proteoform-level pharmacodynamic biomarkers, and reproducible QC for cell therapy and drug programs, filling a critical gap between legacy Westerns and high-bar mass spectrometry and advancing proteoform-centric biology across immunology and oncology.

Layperson summary: Think of DSI-Seq as a modern upgrade to the classic Western blot, the lab test that shows whether a protein is present and how big it is. Instead of a fuzzy picture of bands, it separates proteins by size on a tiny chip, adds simple barcodes, and then uses DNA reading tools to count each protein like a supermarket scanner. The result is a clear table of numbers that tells you not only how much of a protein is there, but also whether it is the full version, a cut piece, or a switched-on form. It can check many proteins at once from a very small sample and gives answers in hours instead of days. This helps scientists see how cells send signals, confirm how drugs and cell therapies work, and compare results across labs with much better consistency. In short, it turns a slow, manual art into a fast, reliable measurement that can speed up discoveries and improve testing in medicine.

Method: Digital Size‑Indexed ImmunoSequencing (DSI‑Seq)

Goal: preserve Western’s size information and antibody specificity, but make it quantitative, multiplexed, fast, and low input.

Core idea
Rapid microchip SDS separation of denatured proteins.
Inline fractionation of the eluting stream into many narrow size bins.

Multiplex DNA‑barcoded immunoassay in each bin.
Sequencing or digital PCR readout to count molecules per target per size bin.

Computational mapping from bin index to molecular weight using co‑run standards.

This yields a 2D matrix: targets on one axis, size bins on the other. You see isoforms and shifts exactly like a blot, but with digital counts instead of gray bands.

Why it beats Westerns

Isoform-resolved, like a blot: size bins let you separate full-length vs cleaved or shifted proteoforms.

High multiplex: 50 to 200 targets per run is realistic with barcoded antibodies.

Quantitative: counts are digital, with internal spike‑ins and UMIs for normalization and linearity.

Low sample: nanoliter fractions and single‑tube chemistry cut input requirements.

Throughput: dozens of samples per day on a benchtop box, no membranes, no overnight incubations.

Reproducibility: fixed microfluidic geometry, DNA barcodes with error correction, built‑in standards.
Workflow overview

Sample prep

Lyse in SDS buffer with protease and phosphatase inhibitors as needed.

Add a small panel of recombinant protein ladder standards spanning 10 to 250 kDa.

Microchip SDS separation

Use a disposable polymer‑sieving microfluidic chip.
Inject sample plus ladder. Separate over a short channel. Total runtime on the order of minutes.

Inline fractionation

At the channel outlet, segment the eluent into nanoliter droplets at a fixed frequency.

Each droplet receives a time‑stamp DNA barcode from a clocked side stream. The time stamp uniquely encodes the size bin because migration time maps to molecular weight. In plate‑based builds, collect sequential fractions across 64 to 128 wells instead of droplets and dispense a unique DNA bin barcode into each well.

Multiplex immunoassay with DNA barcodes

Add a cocktail of antibodies, each conjugated to a unique DNA tag that carries an antibody ID barcode, a unique molecular identifier (UMI), universal priming sites.

Two options for specificity:
Single‑epitope capture: antibodies on magnetic beads capture target in each bin. A reporter oligo is released only upon capture using a proximity‑restricted cleavage or displacement.

Proximity extension assay (PEA): two oligo‑tagged antibodies per target give a ligation‑competent DNA only when both bind the same protein. This strongly reduces off‑target.

The bin time‑stamp barcode is ligated to each target amplicon so every read is labeled with both protein identity and size bin.

Readout

PCR amplify and pool. Quantify by NGS for high plex or by multiplex digital PCR for 5 to 30 targets.

Demultiplex reads to counts per target per bin. Collapse UMIs to remove amplification bias.

Analysis

Fit ladder‑derived calibration to convert bin index to apparent kDa.

Call peaks per target to quantify proteoforms or shifts.

Normalize using: external spike‑in protein standards, per‑bin total protein dye signal, or stable reference proteins.

You can plot fraction profiles the way you read bands now, but with real numbers and confidence intervals.

Key design details

Microfluidics

Short, linear polyacrylamide sieving matrix in a molded chip.

64 to 128 fraction bins across the separation window. Effective resolution comparable to 6 to 12 percent gels.

Droplet mode or 96‑well fractionation module, depending on comfort level.

Barcodes and error control

12 to 16 nt antibody ID barcodes with Hamming distance of 3 or more.

8 to 12 nt time‑stamp barcodes for bins.

8 to 12 nt UMIs to deduplicate PCR bias.

Use a universal primer pair for all targets to simplify amplification.

Antibody conjugation

Protein A/G mediated orientation, then NHS‑PEG‑azide to click a maleimide‑bearing oligo, or use site‑specific conjugation on engineered Fc.

Validate each antibody’s linear epitope binding under 0.05 to 0.1 percent SDS or after SDS quench with cyclodextrin.

Controls

No‑antibody and isotype controls in a few bins to estimate background.

Known‑ratio mixtures of recombinant targets to test linearity.

Phosphatase‑treated lysate as negative control for phospho‑specific panels.

Performance targets to aim for

Multiplex: 100 proteins per reaction with NGS; 10 to 30 with dPCR.

Dynamic range: 4 to 5 orders of magnitude with UMIs and spike‑ins.

Sensitivity: low femtomoles per target per lane is realistic; sub‑microgram total protein per sample.

Size resolution: 5 to 10 percent across 15 to 250 kDa with 64 to 128 bins.

Hands‑on time: minimal, no membranes or overnight incubations.

These are engineering targets, not guarantees. They are chosen based on what each subsystem can credibly deliver.

Validation plan that will convince a hard skeptic

Build the separation plus fractionation stub
Run a prestained ladder through the chip.

Fractionate into 64 bins.
Confirm bin‑to‑kDa mapping with a logistic fit.

Single‑target end‑to‑end test
One antibody with an oligo tag.

Spike recombinant protein at a dilution series into a HeLa lysate.

Show linear counts versus input and a single bin peak at the expected kDa.

Isoform resolution

PARP1 cleavage in apoptosis: treat cells with staurosporine.

Detect 116 kDa full length and 89 kDa cleaved in separate bins.

PTM specificity
Phospho‑ERK1/2: stimulate and phosphatase‑treat controls.

Phospho‑specific antibody should yield bin‑aligned signal that collapses with phosphatase.

Test Multiplex panel

24‑plex T cell signaling panel: CD3ζ pY, ZAP70 pY, LAT pY, SLP76, PLCG1 pY, ERK1/2 pT/pY, AKT pS473, mTOR pS2448, 4EBP1 pT37/46, NF‑κB p65 pS536, beta‑actin as reference.

Compare fold changes to traditional Westerns and phospho‑flow as orthogonal methods.

Reproducibility

10 technical replicates. Compute CV per target per bin.
Batch‑to‑batch chip variation and antibody lot testing.
Practical build choices

Version A: plate‑basedUse capillary electrophoresis into a 96‑well plate, dispense pre‑aliquoted bin barcodes by row, perform the immuno‑DNA chemistry in wells, and read by amplicon sequencing. This is the fastest route with standard gear.

Version B: droplet‑basedMicrofluidic droplet generator assigns the bin time‑stamp barcode in flow, merges with antibody beads, then breaks emulsion for pooled amplification. Higher automation and less loss, but more microfluidic engineering.

Version C: no‑sequencer optionReplace NGS with a panel of TaqMan probes on a digital PCR platform. Lower plex but no dependence on sequencing.

Risks and mitigations

Antibody performance in residual SDSMitigation: dilute SDS below 0.05 percent, use SDS scavengers, favor linear epitope antibodies, or renature briefly before binding.

Carryover between binsMitigation: increase droplet segmentation rate or fraction dead volumes, add a short waste gap between bins, validate cross‑talk with ladder only.

Barcode cross‑talk and index hoppingMitigation: error‑correcting barcodes, unique dual‑indexing, UMI deduplication.

Off‑target binding in complex lysatesMitigation: use PEA two‑antibody logic for problematic targets, include matched isotype and competition controls.

Mapping accuracy from time to kDaMitigation: co‑run ladder and fit per run. Report apparent kDa with confidence intervals.

How it compares to current alternatives

Traditional Western: size info yes, multiplex low, quantitation poor, time long. DSI‑Seq: size info yes, multiplex high, digital counts, faster.

Capillary Western systems: automated and quantitative but limited multiplex. DSI‑Seq: retains automation but scales multiplex via barcodes.

DigiWest‑like bead fractionation: conceptually close but uses protein‑on‑bead plus fluorescent detection. DSI‑Seq: swaps fluorescence for DNA counting with UMIs and a simpler microchip separation.

Targeted mass spectrometry: high specificity and absolute quantitation, but complex setup and limited size visualization. DSI‑Seq: more accessible and preserves a blot‑like picture of proteoforms.

Kit and instrument concept

Disposable chip with sieving matrix and outlet fractionator.

Antibody panel with validated DNA barcodes and UMIs.

Bin barcode plate or droplet side‑stream mix pre‑made.

Calibration ladder proteins plus spike‑in counting controls.

Benchtop controller for voltage, flows, and droplet timing.

Software that auto‑maps bins to kDa, calls peaks, and outputs publication‑ready plots and CSVs.

When denaturation or SDS is unavoidable
If you must process denatured lysate, dilute or scavenge SDS before proximity chemistry. Many Western‑validated antibodies bind linear epitopes after SDS quench.

If two‑epitope binding fails under those conditions, switch the target to Option B capture‑and‑release for that protein while keeping proximity extension for the rest.

Failure modes to plan for, with fixes

High background without protein: incomplete nuclease inactivation or oligo self‑ligation. Fix by tightening the nuclease step and redesigning oligos with higher Hamming distance and blocked ends.

Hook effect at very high abundance: split samples or dilute to keep partition occupancy between 0.1 and 0.7.
Poor agreement between targets: recalibrate conversion efficiencies with purified standards, and check antibody pair compatibility.

Cross‑reactivity: migrate problematic targets to the capture‑and‑release format or require a competition control for acceptance.

Bottom line and cross-check with the landscape:

DSI‑Seq is a novel and feasible direction for a true “Western‑replacement” that preserves size information while giving digital, multiplexed readouts. The closest prior arts solve only parts of this: DigiWest multiplexes after SDS‑PAGE but uses bead fluorescence, not sequencing; Simple Western automates size‑resolved immunoassays but does not scale to high plex; Olink PEA and other DNA‑barcoded immunoassays are highly multiplexed but lack size resolution. Combination of microchip SDS separation + inline fractionation + bin barcoding + DNA‑counting does not appear in the literature as a unified method.

How it compares to what exists
DigiWest: SDS‑PAGE lane is sliced and eluted to barcoded beads, then probed and read by Luminex fluorescence. It retains size info and multiplexes, but detection is not digital sequencing. Your approach swaps in sequencing with UMIs and a microchip separation front‑end.

Simple Western (capillary Western): automated, quantitative, but limited multiplex and no sequencing readout.

DNA‑barcoded immunoassays: Olink PEA and ID‑seq give high plex digital counts by NGS, but they operate in solution without size resolution. You add back the Western’s unique size axis.

Microchip SDS protein sizing: commercially standard with typical ∼10 percent sizing resolution in minutes, which matches your binning targets.

Conclusion on novelty: the integration and the bin time‑stamping idea look patentable and practically differentiating.

Here is the interrogation of feasibility, potential issues problems, how to solve them, improvements to the assay, quality checks, go no go acceptance criteria:

Feasibility verdict

Technically feasible with careful chemistry and controls. The gating risk is background signal from oligo interactions and matrix inhibition of the polymerase. The second risk is biology specific, namely finding antibody pairs that both bind the same protein in lysate while tolerating the buffer you need. Both can be engineered down to acceptable levels with the mitigation stack below.

If your goal is accurate copy numbers for 10 to 20 proteins per sample in standard lysates, this can be achieved. For membrane proteins or harsh buffers, plan to use a bead capture step to remove inhibitors before PCR.

Key risks and how to retire them fast

Antibody binding after SDS Go/no‑go: binding at ≤0.01 percent SDS after cyclodextrin treatment must recover to at least 60–80 percent of native control for a test panel. If not, switch to a rapid buffer‑exchange or on‑bead SDS removal before binding.

Bin cross‑talk Quantify with a fluorescent ladder only. Require <3 percent signal in adjacent bins for the dominant band before moving on.

Barcode cross‑talk and index hopping Use unique dual indexes with error‑correcting antibody ID barcodes. Enforce strict adapter cleanup.

Insufficient effective resolution If dispersion broadens peaks across >3 bins at mid‑range, increase number of bins and shorten the separation window. Note that commercial chips deliver ∼10 percent resolution, which maps well to 64–128 bins over 15–250 kDa.

UMI collisions at high copy bins Use 14–16 nt UMIs and UMI‑aware deduplication tools.

Recommended MVP path

Prioritize Version A, plate‑based. It is the fastest credible build with standard gear.

Step 1. Build separation + fractionation stub
Microchip SDS run into a 96‑well plate. Pre‑aliquot bin barcodes across wells. Confirm bin‑to‑kDa calibration with a prestained ladder.

Acceptance: logistic fit r² ≥ 0.98, sizing error ≤ 10 percent for ladder bands.

Step 2. SDS neutralization check
Titrate β‑cyclodextrin in collected fractions and test one antibody–antigen pair known to be sensitive to SDS. Require ≥ 60 percent of native binding at ≤ 0.01 percent residual SDS.

Step 3. Single‑target end‑to‑end
One DNA‑tagged antibody, HeLa lysate spiked with recombinant target, 8‑point dilution, NGS readout with 16 nt UMIs. Expect linear counts over at least 3 logs with UMI collapse.

Step 4. Isoform resolution
PARP1 cleavage model. Show two non‑overlapping peaks at ∼116 and ∼89 kDa with <10 percent cross‑bin bleed.

Step 5. PTM specificity
ERK1/2 pT/pY with phosphatase control. Phospho signal should vanish in treated samples while total ERK remains. Use PEA for phospho to suppress off‑targets.

Step 6. 24‑plex T‑cell panel
Run your listed phospho panel. Benchmark fold‑changes against Simple Western and phospho‑flow. Expect rank‑order agreement and tighter CVs on DSI‑Seq if normalization is done with spike‑ins and per‑bin protein signal.

Quantitative design notes

Bins and segmentation: with a 2–3 minute separation window and 96–128 bins, you are only asking for ∼0.5–1.0 Hz effective binning. That is easy for a valve or droplet generator, and nanoliter per bin volumes are compatible with standard PCR chemistry. Reviews document CE‑to‑droplet interfaces.

Normalization hierarchy: 1) external spike‑in protein standards, 2) per‑bin total protein measurement via a dye read on the plate before PCR, 3) stable reference proteins.

Library design: error‑correcting 12–16 nt antibody IDs with Hamming distance ≥ 3, 14–16 nt UMIs, and unique dual indexes for samples.

Data analysis: UMI collapse, per‑bin background modeling, then peak calling on smoothed target‑wise bin profiles with FDR control. UMI‑aware tools will handle sequencing errors in UMIs.

Where to be extra hard on yourselves

SDS compatibility is the make‑or‑break. Literature is clear that SDS suppresses immunorecognition in solution. Beta‑cyclodextrin removal is credible, but you must prove quantitative recovery for several antibody classes and for phospho‑epitopes.

True size fidelity. Demand bin‑to‑kDa errors ≤ 10 percent across the ladder. Commercial microchip SDS assays hit this spec, so you should too.

Cross‑method agreement. In the 24‑plex test, require high concordance with Simple Western and targeted MS for several anchors. Targeted MS is the recognized Western alternative for quantitative work. Use it to certify your dynamic range and linearity.

Hard problems you must solve, with fixes

One protein should create one amplicon
Risk: proximity oligos extend or ligate without protein, or multivalent proteins give more than one product.

Fixes:
Use two oligos that are each locked in a short hairpin. A short connector strand opens both hairpins only when co‑localized on the same protein, then a high‑fidelity polymerase fills in to create the amplicon. Include 3′‑phosphate blocking until the connector is present.
For oligomers, design epitope pairs on a single monomer when possible. If not possible, report results per complex rather than per monomer and state that explicitly.

Antibody pair availability and behavior in lysate
Risk: lack of two non‑overlapping linear epitopes, steric clash, or loss of binding in detergent.

Fixes:
Start with clones validated for Western or IP, not only IF. Western and IP clones tolerate linear epitopes and exposure to detergents.

Perform epitope binning by BLI or SPR to choose non‑competing pairs separated by at least 5 to 10 nm on the unfolded or partially refolded protein model.
Where pairs are weak, replace one antibody with a nanobody or aptamer against a distinct linear epitope.
For phospho targets, prefer two antibodies that bind outside the modified residue, with one being modification specific. If this is impossible, move that target to capture‑and‑release.

Matrix inhibition of the proximity and PCR steps
Risk: detergents, salts, or protease inhibitors compromise extension and PCR.

Fixes:
Keep lysis buffer PCR compatible when possible. If you must use strong detergents, run capture‑and‑release: bead capture, wash to remove inhibitors, then release DNA reporter into clean PCR buffer.

Add a short post‑binding buffer exchange on magnetic beads for problematic targets.

Validate a polymerase mix that tolerates residual lysate at your intended dilution.

Background from carryover DNA or oligo self‑assembly
Risk: false positives dominate the low end and inflate the apparent copy number.

Fixes:
Pre‑clear the lysate with Benzonase, then inactivate it completely before proximity chemistry.

Use dUTP in all amplicons and add UDG before PCR to destroy any carryover products from previous runs.
Split pre‑PCR and post‑PCR spaces physically, with aerosol‑barrier tips and separate lab coats.

Sequence design: hairpin locks, high Hamming distance between barcodes, blocked ends, and no complementarity between any non‑partner oligos. Reject any panel where the reagent‑only control exceeds 0.2 positive partitions per 20,000 partitions.

Hook effect and nonlinearity at high abundance
Risk: signal per protein falls at high concentration because the two binders distribute on different molecules.

Fixes:
Titrate antibody concentrations into the zone where both epitopes are saturated for your expected protein range.

Always include a two‑point dilution series per sample for the abundant proteins. Accept a target only if the two dilutions agree within 10 percent after Poisson correction.

Multiplex cross‑talk
Risk: oligos from different pairs hybridize or amplify off target.

Fixes:
Build the panel by subpanels. Screen each new pair against the existing set for off‑target positives in a protein‑free background.

If a pair misbehaves, move it to the capture‑and‑release lane to isolate its chemistry.

Go or no‑go acceptance criteria

Adopt these hard thresholds before you believe a number.

Reagent‑only background
Less than 0.2 positives per 20,000 partitions per target channel.

Blank lysate after nuclease
Less than 0.5 positives per 20,000 partitions with universal primers but without proximity chemistry. If not, your nuclease or physical separation is insufficient.

Spike‑in recovery
Recovery 0.8 to 1.2 across a 100‑fold range for purified protein spiked into matrix. Failures trigger re‑titration of antibody concentrations and buffer cleanup.

Dilution linearity in real lysate
Two dilutions that differ by 2× must report a 1.8 to 2.2 ratio after Poisson correction.

Repeatability
Technical replicate CV less than 10 percent at 400 to 10,000 molecules per reaction.

Cross‑panel interference
Adding or removing other antibody pairs changes the measured copy number by less than 10 percent.

Bead‑gated proximityBind the capture antibody on beads, add the reporter antibody in solution, perform proximity extension on‑bead, then wash and elute the newly formed DNA into PCR buffer. This removes inhibitors and unreacted oligos and cuts background sharply. It reduces hands‑off simplicity but pays off for tough targets.

Connector‑mediated extension with dual locksEncode two short toeholds on the oligo arms and design a connector that bridges both with perfect Tm only when both arms are present. Include 3′ phosphates on arms and a 5′ phosphate on the connector. Use a strand‑displacing polymerase that starts only after ligation or nick‑translation. This reduces unspecific arm‑to‑arm pairing.

UDG carryover suppression and separate primer poolsUse dUTP in all products plus UDG in the PCR master mix. Maintain separate primer mixes for each subpanel to avoid building a universal contaminant that can amplify anywhere.

External protein counting standardInclude a recombinant protein with an artificial two‑epitope tag and its dedicated antibody pair in every reaction at a fixed copy number. This provides an internal process control for conversion efficiency and partition calling. Report all targets relative to this control and then convert back to absolute units with its known copy number.

Per‑target conversion efficiency calibrationMeasure epsilon for each target with a dilution series of purified protein in matrix. Store epsilon in software and apply a simple correction: estimated molecules equal measured molecules divided by epsilon. Update epsilon with each new antibody lot.

Panel design discipline
Keep amplicon length between 60 and 100 nt with matched Tm, design all probes against non‑overlapping internal sequences, and require Hamming distance of at least 3 for barcodes. Reject any oligo that creates primer dimers in silico against the entire panel.

PTM‑aware strategiesFor phosphorylation, when two antibodies cannot both bind, pair a pan antibody with a phospho‑specific binder and place the oligo on the pan arm only. Then use capture‑and‑release where phospho recognition triggers DNA release. This keeps the readout phospho dependent without needing both arms to bind.
Membrane protein handlingFor multi‑pass proteins, use mild detergents such as digitonin or DDM for extraction, then switch to bead capture with washes to remove detergents before the proximity step.

Failure modes you will see first, and how to debug in one day

Everything is positive
Likely contamination with prior amplicon. Replace all solutions, add UDG, and move pre‑ and post‑PCR physically apart. Confirm by running reagent‑only controls.

Nothing is positive, even with recombinant protein
Connector or polymerase step is not working, or one epitope is masked. Verify each arm can be extended alone with a synthetic splint, check antibody binding by classic sandwich ELISA, then return to proximity chemistry.

Strong signal in no‑protein control
Your arms are self‑complementary or the connector bridges unrelated arms. Redesign oligos with hairpins and toeholds that require co‑localization.

Nonlinear response across dilutions
You are in hook territory or conversion efficiency varies with protein concentration. Lower antibody concentration or split the sample. Re‑titrate to achieve flat efficiency across the intended range.

Statistics you should report with every result
Molecules per reaction with 95 percent confidence intervals based on binomial uncertainty of positives, converted via the standard Poisson occupancy model.
Dilution agreement score for each target.

Conversion efficiency epsilon and its uncertainty if you apply the correction.

Background positives per 20,000 partitions for the run.

Practical build choices I recommend

Start plate‑based. Use a microchip SDS instrument or capillary interface to a 96‑well plate, pre‑aliquot bin barcodes, do all chemistry in wells. This gets you data with minimal custom microfluidics. Published interfaces from electrophoresis to droplets or plates give you patterns to copy.

PEA for phospho targets, single‑epitope capture for abundant structural targets. PEA is proven to reduce off‑target noise in multiplex settings.

SDS removal: add β‑cyclodextrin and a brief dilution step upon fraction entry, then bind. Validate residual SDS by a colorimetric assay or mass‑balance against standards.

Controls: isotypes, no‑antibody bins, phosphatase controls for phospho, and known‑ratio recombinant mixes to certify linearity. This mirrors best practices in Western QC and PEA panels.

What you should expect to see if it is working

Distinct, narrow target‑wise profiles in kDa with ladder‑based calibration error ≤ 10 percent over 15–250 kDa.

Linear response over 3–4 logs in single‑target tests after UMI collapse, with per‑bin CV ≤ 15 percent across technical replicates.

In the 24‑plex T‑cell panel, rank‑order agreement with Simple Western and phospho‑flow, with tighter replicate variance when using spike‑ins and total protein normalization.

Read 5 tweets

Derya Unutmaz, MD

@DeryaTR_

Aug 17, 2021

İsrail’in Nisan’da koronayı yenmiş ve toplumsal bağışıklığa ulaştığını düşünmüştük. 2-3 ay boyunca da vaka sayıları 2 haneli rakamlara düştü haftalarca ölüm olmadı. Fakat Temmuz sonunda İsrail’de çok hızlı 4.Covid dalgası başladı, peki ne oldu? Aşılar etkisiz mi ? Bu flood da:

Gerçekten günlük vaka sayıları İsrail’de 4-5 binlere çıktı, şu anda hastanede yatan covid hastaları 500-600 civarı, ölümlerde sıfırdan günlük ortalama 10-20 civarına çıktı, ve bunların yaklaşık yarısı aşılı (çift doz mRNA). Öncelikle, bu yeni dalganın ana sebebi Delta varyantı…/

Delta aslında bir süper varyant, insanlığı enfekte eden en bulaşıcı virüslerden biri oldu. Virüs hücrelere o kadar hızlı giriyor ki bunu durdurmak için çok miktarda akıllı füzenizin hazır bekliyor olması lazım. Yani bir önceki varyantlara yeten antikordan daha fazlası gerekiyor./

Read 10 tweets

Derya Unutmaz, MD

@DeryaTR_

Aug 15, 2021

https://twitter.com/zynpokmen/status/1426568964029698048

Aşı karşıtları & militan takipçilerini anında engelliyorum, lakin bitmek bilmiyorlar, hakaret, saldırı & tehditleriyle…sanki organize çeteler gibi hareket ediyorlar, hayatları pahasına koronayla mücadele eden meslektaşlarımızı tedirgin ediyorlar. Bu kabul edilir bir durum değil.

https://twitter.com/zynpokmen/status/1426568964029698048

https://twitter.com/Cetinn_Ceviz/status/1426807348929105920

Maddi çıkar var tabii, Amerika’da bunların başı Joseph Mercola, geçen yıl sözde takviye “alternatif tıp” ürünlerinden 100 milyon dolar kazanmış! Bu şekilde popüler olan bazı doktorlar da bu işleri yapıyor, tereddütte olan insanları istismar ederek kendi etrafında topluyor.

https://twitter.com/Cetinn_Ceviz/status/1426807348929105920

https://twitter.com/hnfcn/status/1426811632613830656

Çok yazık çok 😞

https://twitter.com/hnfcn/status/1426811632613830656

Read 4 tweets

Derya Unutmaz, MD

@DeryaTR_

Feb 8, 2021

Bugüne kadar koronaya karşı çoğunlukla antikorlardan (akıllı füzelerden) bahsettik ama bağışıklık sisteminin bir de komutanları ve özel kuvvetleri/keskin nişancıları (T hücreleri) var- aslında bunlar benim de 30 yıldır araştırmalarımızın çoğunu adadığım ve en sevdiğim hücreler ++

Bu T hücre komutanları virüsün parçalarını tanımazsa, iyi antikor da oluşmaz. Önemli olarak, akıllı füzelerden kaçıp hücrelere girmeyi başaran virüsleri de T hücre özel kuvvetleri arayıp bulur & yok eder. Güzel tarafı virüs mutasyonla bu T hücrelerinden öyle kolay kaçamaz😊++

Bu yüzden, yeni mutant koronalar aşı olanları daha yüksek oranda enfekte etse de, bu kişiler covid19’u hafif geçiriyorlar-çünkü özel güç T hücreleri virüslerin saklandığı hücreleri bulup yok ediyor ayrıca komutları ile diğer hücreler de alarma geçiriyor. Bağışıklık böyle işte😊

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Derya Unutmaz, MD

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