rAgitoxin-2-Cys-TAMRA
- 貨號 Cat. No.
- RTA-420-T
- 規格 Size
- 1 μg 5x1 μg
Description
rAgitoxin-2-Cys-TAMRA
A Novel Marker of KV1.3 K+ Channels
| Product#: | RTA-420-T |
| Sizes: |
| 1 mg |
| 5 x 1 mg |
| M.W.: | 4673 |
| Sequence: | GVPIN VSCTG SPQCI KPCK C AGMRF GKCMN RKCHC TPK |
 | NMR structure of Agitoxin-2 (1AGT.pdb). Gly1, Thr9, Gly10, Ser11, Arg24, Phe25, Lys 27, Met 29, Asn30 and Thr36 have been found to participate in the interaction with Kv1.3 (marked in green).26 Asp20, located at the opposite face of the protein was replaced with Cys (marked in red, see article).Images were generated using the ViewerLite freeware (Accelrys Inc.). |
| Purity: | >99% |
| Bioassay: |
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1: rAgitoxin-Cys-TAMRA Retains the Inhibitory Characteristics of the Wild Type Toxin A. Kv current inhibition by rAgitoxin-2 (#RTA-420) (100nM, pink) and rAgitoxin-2-Cys-TAMRA (#RTA-420-T) (100nM, blue) in Kv1.3 transfected HEK-293 cells. Top: Time course of Kv1.3 current amplitude is presented. Currents were elicited from holding potential of ?00 mV by 200 ms voltage pulses to +50 mV, every 10 seconds. Periods of toxin perfusions are indicated according to the color code defined above. Bottom: Superimposed traces taken from the same experiment shown on top, before (black) or during toxin perfusion (pink or blue). B. Dose response of Kv1.3 current inhibition in Xenopus oocytes for rAgitoxin-2, rAgitoxin-2-Cys-TAMRA and rAgitoxin-2-Cys (#RTA-420-C). Top: time course of current amplitude, upon toxin perfusion. Currents were elicited from resting potential of ?00 mV, by a 100 ms test pulse to 0 mV, delivered every 10 sec. The bars above the plot represent periods of toxin perfusion, for each toxin sequential applications of the following concentrations: 1, 10, 50 and 100 nM, where the toxin species is indicated. Middle: Superimposed traces representing currents under control conditions (black) and during perfusion of 100 nM toxin:rAgitoxin-2-Cys-TAMRA (orange), rAgitoxin-2 (blue) and rAgitoxin-2-Cys-TAMRA (green). Bottom: Dose response of toxins on current inhibition (n=3 for each toxin, mean and SD). See inset for color coding. |
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2. Kv1.3 Expressing HEK-293 Cells Labeled with rAgitoxin-2-Cys-TAMRA Confocal image of Kv1.3 expressing HEK-293 cells labeled with a fluorescent rAgitoxin-2-Cys-TAMRA (#RTA-420-T). Kv1.3 expressing HEK-293 cells were grown on glass cover-slips, incubated in the presence of 50nM rAgitoxin-2-Cys-TAMRA for 30 minutes at room temperature and washed with PBS. Left: Bright light field image of the cells. Middle: Image acquired with a Rodamine filter. Right: Merge of both images. As can be seen, rAgitoxin-2-Cys-TAMRA is localized with the membrane-anchored Kv1.3 K+ channel. We would like to thank Mr. Vladimir Kiss and Dr. Noga Alagem from the Weizmann Institute of Science for their help with the confocal microscope images. |
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3. rAgtitoxin-2-Cys-TAMRA Binding to HEK Cells Stably Transfected with Kv1.3 Channels. Top: Staining of HEK-293 cells stably expressing Kv1.3 channels with 50 nM of rAgitoxin-2-Cys-TAMRA (#RTA-420-T). Cells were incubated for 30 minutes with the fluorescent toxin, washed with PBS and visualized. In the middle section the image acquired with a Rodamine filter is shown and on the right it is merged with the bright light image of the same field to illustrate cellular staining. Middle: Displacement of labeled toxin by rAgitoxin-2 (#RTA-420). The same preparation as the one shown on top was pre-incubated for 30 minutes with 10 然 of rAgitoxin-2, followed by another 30 minute incubation after addition of 50 nM rAgitoxin-2-Cys-TAMRA. Bottom: rAgitoxin-2-Cys-TAMRA does not stain non-transfected HEK-293 cells. The procedure was as in the top panel. |
| References: |
1. | |
2. | Cayabyab, F.S. et al. (2000) Eur. J. Neurosci. 12, 1949. |
3. | Hafidi, A. et al. (2005) Neuroscience 130, 475. |
4. | Freudenthaler, G. et al. (2002) Histochem. Cell Biol. 117, 197. |
Native Agitoxin-2 was originally isolated from the venom of the Israeli scorpion L. quinquestriatus hebraeus.1 Agitoxin-2 inhibits the native Kv1.3-like current in cultured microglia and Kv1.3 in MLS-9 cells.2 rAgitoxin-2 is a potent blocker of the Shaker voltage-gated K+-channels as well as the mammalian homologues of Shaker.1
A few recombinant, mutated and labeled toxins have been described in the literature, including the KCa1.1 specific blocker Iberiotoxin (#RTI-400) and the KV1 blocker Hongotoxin (#RTH-400). 3-4
A similar approach was taken by Alomone Labs in producing rAgitoxin-Cys-TAMRA. A fluorescent dye is attached to a free Cys residue, which replaced Asp in position 20 of the 38 amino acid long peptide. This novel toxin retained the blocking activity of the original toxin and was demonstrated to specifically label living cells expressing KV1.3 channels.