Vitcylation Probes

Ethyl-Vitcylation Probe

A cell-permeable chemical probe for detecting protein Vitcylation modifications in living cells. The ethyl ester design enhances membrane permeability, enabling intracellular labeling and real-time monitoring of Vitcylated proteins via Click Chemistry.

Product Information

Cat. No.Vitcyl-001

Concentration500 mM

Pack Size10 μL / 100 μL

Storage−80 °C, dark

UseResearch Only

Applications

Intracellular Vitcylation regulator screening Post-translational modification (PTM) analysis Fluorescence scanning (SDS-PAGE) Live-cell Vitcylation detection

Experimental Protocol

Cell Preparation

Seed cells in a 12-well plate. Culture to 70–80% confluency.

Probe Labeling

Remove medium, wash with PBS. Add 500 μL fresh medium containing Ethyl-Vitcylation Probe.

Final: 500 μM • 37 °C • 2 h

Protein Extraction

Wash with PBS (×2). Lyse with 100 μL 1% NP-40 lysis buffer (with protease inhibitors). Vortex 1 min, ice 10 min, repeat ×3. Centrifuge 12,000 rpm, 4 °C, 10 min.

Click Chemistry Labeling

Mix: 1 μL CuSO₄ + 1 μL BTTAA → add 1 μL 5-TAMRA-N₃ + 2 μL sodium ascorbate. Add 5 μL Mix to 35 μL protein sample.

37 °C • 30 min • dark

SDS-PAGE

Add loading buffer, heat 95 °C for 10 min. Load 10 μL on 8–10% gel and run electrophoresis.

Fluorescence Detection

Wash gel with ddH₂O. Scan at 532 nm / Cy3 channel (e.g. Amersham Typhoon™). Stain with Coomassie Blue for loading normalization.

Click Reaction System — Single Sample

Component	Stock Conc.	Amount	Solvent	Added Volume	Final Conc.
Sample (Protein Lysate)	—	—	—	35 μL	—
5-TAMRA-N₃	3 mM	5 mg	DMSO	1 μL	75 μM
CuSO₄ · 5H₂O	40 mM	20 mg	H₂O	1 μL	1 mM
BTTAA	80 mM	70 mg	DMSO	1 μL	2 mM
Sodium Ascorbate	40 mM	16 mg	H₂O	2 μL	2 mM
Buffer Supplement	—	—	H₂O	Adjust to 40 μL	—

Representative Result

Cell treatment (HeLa)
Probe (mM): 0 / 0.2 / 0.5

Vitcylation

Coomassie

Ethyl-Vitcylation Probe
Dose-dependent signal

Dose-Dependent Vitcylation Signal in HeLa Cells

HeLa cells were treated with Ethyl-Vitcylation Probe at 0, 0.2, and 0.5 mM for 2 hours. Following Click Chemistry labeling and SDS-PAGE analysis, fluorescence scanning revealed progressively increased Vitcylation signals with rising probe concentration.

Coomassie staining confirmed comparable protein loading across all lanes.

Vitcylation signal intensity increases proportionally with probe concentration, validating dose-responsive intracellular labeling in living cells.

Storage & Precautions

❄ Storage Conditions

Store at −80 °C, protected from light
Avoid repeated freeze-thaw cycles
Aliquot before use whenever possible

⚠ Precautions

Click Chemistry requires a freshly prepared catalytic system
All procedures must be protected from light
For research use only

Vitcylation Probe

A chemical probe designed for detecting protein Vitcylation modifications in cell lysate systems. Following covalent reaction with proteins, fluorescent labels are introduced via Click Chemistry, enabling validation of Vitcylated proteins and modification sites in vitro.

Product Information

Cat. No.Vitcyl-002

Concentration500 mM

Pack Size10 μL / 100 μL

Storage−80 °C, dark

UseResearch Only

Applications

Vitcylated protein validation Modification site identification In vitro Vitcylation verification LC-MS enrichment workflows

Experimental Protocol

Cell Collection

Seed cells in a 6-well plate to 70–80% confluency. Collect by trypsinization or direct scraping. Wash with PBS (×3) and transfer to EP tube.

Protein Extraction

Lyse with 100 μL 2% SDS lysis buffer (with protease inhibitors). Vortex 1 min, ice 10 min, ×3. Add 0.1 μL nuclease, 37 °C 30 min. Centrifuge 12,000 rpm, 10 min.

SDS Dilution

Add 400 μL PBS to dilute SDS to ≤0.4%. SDS above 0.4% significantly inhibits Click reaction efficiency.

⚠ SDS ≤ 0.4% is critical

Probe Labeling

Divide into two groups (100 μL each). Case: add probe to 500 μM. Negative control: add equal volume DMSO.

37 °C • 1,000 rpm • 3 h • dark

Click Chemistry Labeling

Prepare Click Mix: CuSO₄ + BTTAA → 5-TAMRA-N₃ + sodium ascorbate. Add 5 μL Mix to 35 μL protein sample.

37 °C • 30 min • dark

SDS-PAGE & Detection

Denature at 95 °C, 10 min. Load 10 μL on 8–10% gel. Scan at 532 nm / Cy3 channel. Stain with Coomassie Blue.

Click Reaction System — Single Sample

Component	Stock Conc.	Amount	Solvent	Added Volume	Final Conc.
Sample (Protein Lysate)	—	—	—	35 μL	—
5-TAMRA-N₃	3 mM	5 mg	DMSO	1 μL	75 μM
CuSO₄ · 5H₂O	40 mM	20 mg	H₂O	1 μL	1 mM
BTTAA	80 mM	70 mg	DMSO	1 μL	2 mM
Sodium Ascorbate	40 mM	16 mg	H₂O	2 μL	2 mM
Buffer Supplement	—	—	H₂O	Adjust to 40 μL	—

Representative Result

In vitro (HeLa lysate)
Probe: − / +

Vitcylation

Coomassie

Vitcylation Probe
0.5 mM · 3 h

Stable Vitcylation Signal in HeLa Cell Lysate

HeLa cell lysates treated with Vitcylation Probe (0.5 mM) for 3 hours generated robust fluorescence signals following Click Chemistry labeling and SDS-PAGE analysis.

Coomassie staining confirmed comparable protein loading between treated and control groups.

Vitcylation Probe reliably labels Vitcylated proteins in cell lysate systems, providing a consistent signal for in vitro mechanistic and discovery studies.

Storage & Precautions

❄ Storage Conditions

Store at −80 °C, protected from light
Avoid repeated freeze-thaw cycles
Aliquot before use whenever possible

⚠ Precautions

Suitable for in vitro (cell lysate) systems only
SDS in reaction system must be ≤ 0.4%
Click Chemistry requires freshly prepared catalytic system
All procedures must be protected from light
For research use only

Vitcylation Research Probes