Overview
Alternative Name ω-agatoxin-Aa4a, ω-AGTX-Aa4a, ω-Aga-IVA, ω-agatoxin-4A
Lyophilized Powder yes
Origin Agelenopsis aperta (North American funnel-web spider).
Source Synthetic modified peptide
MW: ~6144 Da
Form Lyophilized from double distilled water (ddH2O). May contain TFA as a residual counter ion.
Effective concentration 0.5 – 3 µM
Sequence KKKCIAKDYGRCKWGGTPCCRGRGCICSIMGTNCECKPRLIMEGLGLA-OH
Modifications Disulfide bonds between: Cys4-Cys20, Cys12-Cys25, Cys19-Cys36 and Cys27-Cys34
ATTO Fluor-647N
ATTO Fluor-647N
Label ATTO-647N. ATTO dyes are characterized by strong absorption (high extinction coefficient), high fluorescence quantum yield, and high photo-stability. Maximum absorption 646 nm; maximum fluorescence 664 nm. The fluorescence is excited most efficiently in the range 625 - 660 nm. A suitable excitation source is the 647 nm line of the Krypton-Ion laser or a diode-laser emitting at 650 nm. It can be used in flow cytometry (FACS) using the red (637 nm) laser. The extent of labeling is 1-2 molecules of dye per ω-Agatoxin IVA molecule.
Structure
Activity ω-Agatoxin IVA is a blocker of voltage-sensitive P-type Ca2+ channels1. It blocks neuromuscular transmission presynaptically in a variety of synapses2,3.
References-Activity
- Mintz, I. et al. (1992) Nature 355, 827.
- Meir, A. et al. (1999) Physiol. Rev. 79, 1019.
- Stephens, G.J. et al. (2001) Eur. J. Neurosci. 13, 1902.
Accession number P30288
Shipping and storage The product is shipped as a lyophilized powder at room temperature. Upon receipt, store the product at -20°C. Protect from moisture. Avoid exposure to light.
Solubility The product is lyophilized in 0.5 ml conical vial.
Centrifuge the vial before adding solvent (10,000 x g for 5 minutes). The lyophilizate may be difficult to visualize. Add solvent directly to the centrifuged vial. Tap the vial to aid in dissolving the lyophilized product. Tilt and gently roll the liquid over the walls of the vial. Avoid vigorous vortexing. Light vortexing for up to 3 seconds is acceptable if needed.
Soluble in pure water at high-micromolar concentrations (50 µM - 1 mM). 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 100-1000x of the final working concentration. Divide the stock solution into small aliquots and store at -20°C. Before use, thaw the relevant vial(s) and dilute to the desired working concentration in your working buffer. Centrifuge all product preparations at 10,000 x g for 5 minutes before use. It is recommended to prepare fresh solutions in working buffers just before use. Avoid multiple freeze-thaw cycles to maintain biological activity. Avoid exposure to light.
Centrifuge the vial before adding solvent (10,000 x g for 5 minutes). The lyophilizate may be difficult to visualize. Add solvent directly to the centrifuged vial. Tap the vial to aid in dissolving the lyophilized product. Tilt and gently roll the liquid over the walls of the vial. Avoid vigorous vortexing. Light vortexing for up to 3 seconds is acceptable if needed.
Soluble in pure water at high-micromolar concentrations (50 µM - 1 mM). 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 100-1000x of the final working concentration. Divide the stock solution into small aliquots and store at -20°C. Before use, thaw the relevant vial(s) and dilute to the desired working concentration in your working buffer. Centrifuge all product preparations at 10,000 x g for 5 minutes before use. It is recommended to prepare fresh solutions in working buffers just before use. Avoid multiple freeze-thaw cycles to maintain biological activity. Avoid exposure to light.
Storage of solutions Store the reconstituted solution at -20°C for the shortest time possible. Avoid multiple freeze-thaw cycles. We do not recommend storing the product in working solutions for longer than a day. Avoid exposure to light.
Our bioassay
Live cell imaging of ω-Agatoxin IVA-ATTO Fluor-647N in differentiated PC-12 cells.Neurite outgrowth was induced in PC-12 cells through 8 days exposure to 100 ng/ml Native mouse NGF 2.5S protein (99%) (#N-240). (A) CellMask™ Actin 1X solution was applied for 30 minutes, resulting in a green fluorescence to visualize cellular membrane. (B) Following this, the same cells underwent incubation with 0.3 µM of ω-Agatoxin IVA-ATTO Fluor-647N (#STA-500-FRN) for 60 minutes at 37ºC, followed by PBSX1 wash, leading to red fluorescence indicative of the distribution of CaV2.1 channels. (C) Live imaging of the differentiated PC-12 cells allowed observation of ω-Agatoxin IVA-ATTO Fluor-647N distribution among the cells.
Unlabeled ω-Agatoxin successfully blocks CaV2.1 channels access.Neurite outgrowth was induced in PC-12 cells through 8 days exposure to 100 ng/ml Native mouse NGF 2.5S protein (99%) (#N-240). (A) CellMask™ Actin 1X solution was applied for 30 minutes, resulting in a green fluorescence to visualize cellular membrane. (B) Following this, the same cells underwent incubation with 100 µM of ω-Agatoxin IVA (#STA-500) and 0.3 µM of ω-Agatoxin IVA-ATTO Fluor-647N (#STA-500-FRN) for 60 minutes at 37ºC, followed by PBSX1 wash. (C) Live imaging of differentiated PC-12 cells demonstrates that ω-Agatoxin IVA successfully competes with ω-Agatoxin IVA-ATTO Fluor-647N for binding sites of CaV2.1 channels.
ω-Agatoxin IVA toxin co-localizes with presynaptic Anti-Neuroligin 1 (NLGN1) in mouse Cerebellum.(A) Fixed mouse brain sections were incubated with ω-Agatoxin IVA-ATTO Fluor-647N (#STA-500-FRN), 0.5 µM, for 1h at 37ºC. (B) Same sections were stained with Anti-Neuroligin 1 (extracellular) Antibody (#ANR-035), (1:300), followed by goat-anti-rabbit-AlexaFluor-568 (seen in green). (C) Cell nuclei are stained with DAPI (blue). (D) Merge of A-C shows co-staining of ω-Agatoxin IVA-ATTO Fluor-647N, and NLGN1 (arrows) in white matter axons.
ω-Agatoxin IVA toxin stains mouse Cerebellum.(A) Fixed mouse brain sections were incubated with ω-Agatoxin IVA-ATTO Fluor-647N (#STA-500-FRN), 0.5 µM, for 1h at 37ºC. (B) Cell nuclei are stained with DAPI (blue). (C) ω-Agatoxin IVA-ATTO Fluor-647N stains Purkinje cells (arrows).
Direct flow cytometry of ω-Agatoxin IVA in live intact rat PC-12 cells.___ PC-12 cells..
___ PC-12 cells + 0.5 µM ω-Agatoxin IVA (#STA-500).
___ PC-12 cells + 0.5 µM ω-Agatoxin IVA-ATTO Fluor-647N (#STA-500-FRN).
Alomone Labs ω-Agatoxin IVA-ATTO Fluor-647N inhibits CaV2.1 channel currents heterologously expressed in Xenopus oocytes.A. Representative time course of ω-Agatoxin IVA-ATTO Fluor-647N (#STA-500-FRN) inhibition of CaV2.1 channels current. Membrane potential was held at -100 mV, current was elicited by a 100 ms voltage ramp to +60 mV every 10 sec, and inhibited by 2.5 µM ω-Agatoxin IVA-ATTO Fluor-647N (green).
B. Superimposed traces of CaV2.1 currents after application of control (black) and of 2.5 µM ω-Agatoxin IVA-ATTO Fluor-647N (green), taken from the recording in A.
References - Scientific background
- Mintz, I.M. et al. (1992) Nature 355, 827.
- Moreno, H. et al. (1997) Proc. Natl. Acad. Sci. 94, 14042.
- Bourinet, E. et al. (1999) Nat. Neurosci. 2, 407.
- Meir, A. et al. (1999) Physiol. Rev. 79, 1019.
- Stephens, G.J. et al. (2001) Eur. J. Neurosci. 13, 1902.
- Berrow, N.S. et al. (1997) Eur. J. Neurosci. 9, 739.
- Pearson, H.A. et al. (1995) J. Physiol. 482, 493.
- Nakanishi, S. et al. (1995) J. Neurosci. Res. 41, 532.
Scientific background
Native ω-Agatoxin IVA (ω-Aga-IVA) was originally isolated from Agelenopsis aperta spider venom, and was shown to be a selective blocker of CaV2.1 (P/Q type) channels1. However, the sensitivity depends on the auxiliary b subunit isoform2 and on the splice variant3. Therefore, the effective concentration varies between systems. In accordance, the toxin blocks presynaptic Ca2+ currents and synaptic transmission in a variety of synapses4,5.
ω-Agatoxin IVA is widely used in electrophysiological measurements of cloned and native channels6,7. It is used to assess the role of CaV2.1 channels in synaptic transmission4. In addition, it was used to map the spatial distribution of CaV2.1 channels in mouse cerebellar and hippocampal brain slices8.
Target P-type Ca2+ channels
Lyophilized Powder
ω-Agatoxin IVA-ATTO Fluor-647N (STA-500-FRN) is a highly pure, synthetic, and biologically active conjugated toxin.
Benefits of ω-Agatoxin IVA-ATTO Fluor-647N:
✓ Localization and distribution
✓ Clustering and internalization kinetics
✓ Live cell imaging
✓ Single cell detection
✓ Binding kinetics
✓ Direct flow cytometry
We gladly take on collaboration projects. Please Contact Us.
For research purposes only, not for human use
Last Update: 25/11/2024
Applications
Citations
Citations
Powered by Bioz
