Overview
Lyophilized Powder yes
Origin Synthetic modified peptide
MW: 2601 Da
Effective concentration 1-50 nM
Sequence CNCKAPETALCARRCQQH-NH2
Modifications His18 - C-terminal amidation
Disulfide bonds location- Cys1-Cys11, and Cys3-Cys15
Disulfide bonds location- Cys1-Cys11, and Cys3-Cys15
Label ATTO Fluor-488. ATTO dyes are characterized by strong absorption (high extinction coefficient), high fluorescence quantum yield, and high photo-stability. ATTO-488 maximum absorption is 501 nm and maximum fluorescence is at 523 nm. The fluorescence is excited most efficiently in the 480 - 515 nm range. This label is analogous to the well-known dye fluorescein isothiocyanate (FITC) and can be used with filters typically used to detect FITC. The extent of labeling is 1-molecule of dye per Apamin molecule.
Structure
Activity Apamin is a blocker of voltage-independent Ca2+-activated K+ (SK) channels1.
Shipping and storage The product is shipped as a lyophilized powder at room temperature. Upon receipt, the lyophilizate should be stored at -20°C. Protect from moisture. Protect from light and moisture.
Solubility Centrifuge all products BEFORE adding solvent (10,000 x g for 5 minutes). The preparation of fresh solutions in working buffers before use is recommended.
Soluble in pure water to high-micromolar concentrations (50µM-1mM). For long-term storage in solution, it is recommended to prepare a stock solution by dissolving the product in double distilled water (ddH2O) at a concentration between x100-1000 of the final working concentration. Divide the solution into single-use aliquots and store at -20°C. Before use, thaw the relevant vial(s) intended for use and dilute in the desired working buffer. Avoid multiple freeze-thaw cycles to maintain toxin activity.
Soluble in pure water to high-micromolar concentrations (50µM-1mM). For long-term storage in solution, it is recommended to prepare a stock solution by dissolving the product in double distilled water (ddH2O) at a concentration between x100-1000 of the final working concentration. Divide the solution into single-use aliquots and store at -20°C. Before use, thaw the relevant vial(s) intended for use and dilute in the desired working buffer. Avoid multiple freeze-thaw cycles to maintain toxin activity.
Storage of solutions Avoid exposure to light. Store the reconstituted solution for the shortest time possible at -20°C. We do not recommend storing the product in working solution for longer than one day. Avoid multiple freeze-thaw cycles.
Our bioassay
Apamin toxin binding sites co-localize with SK1, and SK2 ion channels in mouse Thalamus.(A) Fixed mouse brain sections were incubated with Apamin ATTO Fluor-488 (STA-200-AG), 0.8 µM. (B-C) Same sections were stained with Anti-KCNN1 (KCa2.1, SK1) Antibody (#APC-039), (1:100), and Anti-KCNN2 (KCa2.2, SK2) Antibody (#APC-028-GP), (1:100), followed by goat-anti-rabbit-AlexaFluor-568 (yellow), and goat-anti-guinea pig-AlexaFluor-647 (red). (D) Merge of A-C shows co-staining of Apamin ATTO Fluor-488, SK1, and SK2 (arrows) in thalamus neurons. Cell nuclei are stained with DAPI (blue).
Live cell imaging of Apamin-ATTO Fluor-488 in differentiated PC-12Neurite outgrowth was induced in PC12 cells through a four-day exposure to 100 ng/ml Native mouse NGF 2.5S protein (>95%) (#N-100). (A) CellMask™ Actin 1X solution was applied for 30 minutes, resulting in a dark orange fluorescence to visualize cellular morphology. (B) Following this, the same cells underwent incubation with 3 µM of Apamin-ATTO Fluor-488 for 60 minutes at 370C, followed by PBSX1 wash, leading to green fluorescence indicative of the distribution of SK channels. (C) Live imaging of the differentiated PC-12 cells allowed observation of Apamin distribution among the cells, while cell nuclei were visualized using Hoechst 33342 staining at a concentration of 5 µg/ml, emitting blue fluorescence.
Direct flow cytometry of Apamin in live intact rat PC-12 cells.___ PC-12 cells.
___ PC-12 cells + 1 µM Apamin (#STA-200).
___ PC-12 cells + 1 µM Apamin-ATTO Fluor-488 (#STA-200-AG).
*Apamin-ATTO Fluor-488 was incubated at 370C for 60 min.
Apamin-ATTO Fluor-488 blocks rat SK2 channels stably transfected in HEK293T cells.Currents were measured in 293T cells stably expressing SK2 channels using whole cell voltage clamp in presence of Ca2+ in the pipette solution. A 150ms voltage ramp from -120mV to +60mV was applied every 10sec (holding potential -80mV, no leak subtraction). A. Time course at 0mV, showing the effect of 1nM and 10nM. B. Representative current traces at control conditions and following 120s application of 1nM and 10nM (as indicated).
Scientific background
Apamin is a natural peptide isolated and purified from Apis mellifera bee venom. Apamin blocks small conductance Ca2+-activated K+ channels (SK). It is specific for the SK1-3 isoforms, but ineffective in blocking other calcium activated K+ channels2. In mammalian cell lines, Apamin blocks expressed hSK1 with an IC50 of ~10 nM3, rSK1 and rSK2 with IC50 of ~3 and <1 nM respectively4 and liver rSK3 with IC50 <1 nM.5 In Jurkat T-cells, 5 nM of Apamin blocks 70% of hSK2 currents.6
Blocking these channels with Apamin slows down neuronal activity after hyperpolarization7, as well as smooth muscle contraction8 and catecholamine secretion in adrenal glands.9 Electrophysiology application of long (~400 ms) voltage ramps, covering the whole physiological range (-160 to +40 mV), in the whole cell patch-clamp or voltage clamp configurations, before and after bath perfusion of the drug, allows the detection of the blocked channel currents (by comparison).1
Target KCa2 K+ channels
Lyophilized Powder
For research purposes only, not for human use
Last Update: 16/07/2024
Specifications
Citations
Citations
