Using Native Gel Electrophoresis and Phosphofluoroimaging to Analyze GFP-Tagged Proteasomes
Bibliographic record
Abstract
Native polyacrylamide gel electrophoresis (PAGE) is an invaluable technique in biochemistry to characterize native protein complexes with high molecular mass. Thus, native PAGE is suited to resolve proteasomes, giant proteases responsible for the degradation of polyubiquitylated proteins. Proteasomes contain multiple subunits and exist in different configurations. All configurations have a common 20S core particle (CP). The CP encloses the proteolytic chamber and is composed of four stacked rings with C2 symmetry. The entrance to the CP is gated by central pores within the outer rings, which also provide the binding sites for the 19S regulatory complex (RP). Adjacent regulatory proteins, such as Blm10/PA200, are bound to specific proteasome species of low abundance and contribute to the heterogeneity of proteasome complexes. To get insight into the complexity of proteasome configurations in yeast, we developed a native PAGE system by which GFP-labelled variants of proteasomal subunits are visualized by phosphofluoroimaging. Following native PAGE, proteasome species can be subjected to in-gel activity assays, subsequent SDS-PAGE, and Western blotting.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".