A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis
Bibliographic record
Abstract
Reversible phosphorylation is a key signaling mechanism for modulating the functional properties of proteins involved in numerous cellular events. Abnormal protein phosphorylation causes many human diseases. Experimental procedures for the determination of the phosphorylation status of certain proteins are therefore very important in relation to studies on a diverse range of physiological and pathological processes. We have previously reported a novel phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique using a dizinc(II) complex of the phosphate-binding ligand Phos-tag in conjunction with a neutral-pH gel system to detect shifts in the mobilities of phosphoproteins (Zn2+–Phos-tag SDS-PAGE). However, this handmade gel-based procedure is often laborious and time-consuming to perform, and requires a skillful analyst. More recently, SuperSep Phos-tag precast gel has been developed on the basis of the Zn2+–Phos-tag SDS-PAGE methodology. This novel ready-to-use system employs a neutral-pH gel containing 12.5% (w/v) polyacrylamide and the immobilized Zn2+–Phos-tag (50 µM), which is generally used in conjunction with a Tris–glycine-based electrophoretic running buffer. We examined the potential usage of a Tris–N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tris–Tricine) buffer as an alternative running buffer for the SuperSep Phos-tag precast gel system in the analysis of low-molecular-mass phosphoproteins. Compared with Tris–glycine, the Tris–Tricine running buffer improved the resolution of 8.8-35 kDa phosphoproteins and phosphopeptides. We can therefore provide a laborsaving, timesaving, and more reliable strategy for separation of low-molecular-mass phosphoproteins in Phos-tag affinity electrophoresis.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".