Domain: Natural Science — Biochemistry & Molecular Biology
Target concept: Ni-NTA agarose column binding to blue His-tagged protein, followed by elution

Video generation prompt: A vertically oriented Ni-NTA agarose column was loaded with the blue His-tagged protein, followed by washing and elution as the buffer flowed through and was collected.

Evaluation guide:
(i) Target concept: Affinity chromatography using Ni-NTA resin to isolate His-tagged protein, visualized by intrinsic blue color. Minimum mechanism: (a) His-tag binds Ni²⁺ only in loading buffer (low imidazole); (b) washing removes unbound/non-specific proteins in same buffer; (c) elution requires competitive displacement by imidazole, releasing *only* bound His-protein → blue reappears exclusively in eluate. (ii)Storyline: [Frame 0–2] Blue liquid poured into top of vertical column → [Frame 3–5] Colorless buffer flows through column into waste (no collection shown) → [Frame 6–8] Elution buffer begins flowing; column outlet stream remains colorless until resin bed interface is reached → [Frame 9–10] Blue stream emerges and enters stationary collection container. Perspective: Fixed frontal view, full column in frame, outlet centered, collection container directly below, no occlusion. Visibility: Resin bed must be visibly granular and static; blue color must be unambiguously localized to liquid phase (not column wall or air).

Manual:
Experimental Procedure
1. Load blue His-tagged protein solution onto top of vertically oriented Ni-NTA agarose column.
2. Wash column with buffer: colorless effluent flows through and is discarded.
3. Elute bound protein with imidazole-containing buffer: blue eluate flows out and is collected.
Observation of Experimental Phenomena
- Blue liquid enters column top → disappears during washing (colorless outflow) → reappears selectively in collected fraction during elution.
- Column maintains packed granular resin bed; buffer flows continuously top-to-bottom; collected eluate is blue only during elution phase.
Summary of Experimental Principles
Ni²⁺ ions immobilized on agarose chelate His-tags; blue color indicates presence of His-tagged protein; imidazole competes for Ni²⁺ binding, releasing purified protein.

Prompt Grounding rubric:
level_1: No vertical column appears; no blue liquid is shown at any point; no buffer flow through the column is depicted; no collection container appears; none of these four elements (column, blue liquid, flowing buffer, collection container) are present.
level_2: At least one element (a column, or some blue liquid) appears, but the column is not shown upright; there's no visible sequence of loading, buffer flowing, then something being collected; the buffer stream and the collection container are not connected to the column's outlet.
level_3: An upright, vertically oriented column packed with a visible granular resin bed is shown; blue liquid is added to the top of the column at some point; buffer visibly flows down through the column; liquid is visibly collected in a container placed below the column's outlet; loading, buffer flow, and collection each appear as distinguishable stages in the video.
level_4: Everything in score 3 still applies, with the added condition that the column stays upright and fully in frame the entire time with no hands, labels, or unrelated tubes blocking the column-to-outlet-to-container path, and the flow is unambiguously downward from top to bottom with no pooling or backflow at the outlet.
level_5: Everything in score 4 still applies, with the added condition that the blue liquid is added to the column only once, at the very start of the video, the column's outlet, the falling buffer stream, and the collection container line up along a single straight vertical path visible in every frame, and there is no visual glitch, flicker, or rendering break in the buffer stream or resin bed at any point.

Scientific and Causal Correctness rubric:
level_1: The blue color shows up in every stage (loading, washing, and elution alike) or never shows up at all; the buffer appears to flow upward or sideways instead of down through the column; the collected liquid is colorless when it should be blue, or blue when it should be colorless; there is no visible cause-and-effect between the buffer flowing and the blue color appearing or disappearing.
level_2: Blue color shows up during loading and again during collection, but it also lingers through the washing stage instead of disappearing; or blue appears in the collected fraction with no loading stage ever shown; or the column shows no resin bed at all (looks like an empty tube or a solid block); or liquid is being collected before any buffer has flowed through the column.
level_3: Blue liquid is loaded into the column; during washing, what flows out is colorless and is not shown being collected; during elution, blue reappears and is shown being collected; this loading-colorless-wash-then-blue-elution sequence matches how affinity binding, washing, and elution actually work; the column shows a visibly granular, packed resin bed consistent with an agarose matrix.
level_4: Everything in score 3 still applies, with the added condition that the blue color in the collected fraction only starts appearing after the washing stage has fully ended (no overlap between the two), the effluent leaving the column during washing stays completely colorless the whole time, the resin bed stays put and doesn't disperse, dissolve, or float upward during the flow, and the blue color of the eluate matches the blue of the elution buffer with no shift toward green or purple.
level_5: Everything in score 4 still applies, with the added condition that not a single frame of the washing effluent shows any blue tint, the first appearance of blue in the collected eluate lines up with the moment elution buffer first reaches the resin bed (no noticeable lag), the blue color of the collected eluate looks like a similar strength of blue to what was originally loaded rather than a washed-out pale tint, and no blue color appears to originate from somewhere other than the column's outflow (like the air, the tubing, or the column wall).

Spatiotemporal Consistency rubric:
level_1: The column jumps to a different spot or vanishes between frames; blue liquid appears or disappears with no visible source pouring it in or drain taking it away; the falling stream breaks into disconnected droplets that don't follow a continuous path; the collection container changes position or angle partway through; the shot's lighting or shadows shift abruptly as if the scene had been re-shot.
level_2: The column tilts or rotates more than about 10 degrees off vertical between frames; the buffer stream visibly breaks or suddenly changes width/opacity with no explanation; the collection container only appears once elution starts rather than being in place beforehand; the resin bed visibly sinks or compresses while buffer is flowing.
level_3: The column stays upright with no rotation or drifting; the buffer flows as one unbroken stream from the outlet down to the collection container; the collection container stays in the same spot and fully visible before, during, and after elution; the resin bed's height and grainy texture stay consistent; the lighting on the column and container looks the same from the start of the video to the end.
level_4: Everything in score 3 still applies, with the added condition that the stream keeps a consistent thickness and opacity all the way from the outlet to where it lands, the camera's viewpoint doesn't jump or shift perspective partway through, the column/outlet/stream/container all stay in the same relative arrangement in every frame, and the resin bed's grains don't flicker in and out of view.
level_5: Everything in score 4 still applies, with the added condition that the stream follows one smooth, unbroken path from the outlet to the liquid surface with no kinks or reversals, the stream only disturbs the surface of the collected liquid at the point of contact (no rippling or splashing before it actually lands), no blur ever hides the column, the resin bed, or the boundary between colored and colorless liquid, and no frame repeats or skips in a way that makes the flow look jumpy.