- Alomone Labs Agitoxin-2-Cys-TAMRA retains the inhibitory characteristics of the wild type toxin.A. KV current inhibition by Agitoxin-2 (#STA-420), (100nM, pink) and Agitoxin-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 -100 mV by 200 ms voltage pulses to +50 mV, every 10 seconds. 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 Agitoxin-2, Agitoxin-2-Cys-TAMRA and Agitoxin-2-Cys. Top: time course of current amplitude, upon toxin perfusion. Currents were elicited from resting potential of -100 mV, by a 100 ms test pulse to 0 mV, delivered every 10 seconds. 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: Agitoxin-2-Cys-TAMRA (orange), Agitoxin-2 (blue) and Agitoxin-2-Cys (green). Bottom: Dose response of toxins on current inhibition (n = 3 for each toxin, mean and SD). See inset for color coding.
- Alomone Labs Agitoxin-2-Cys-TAMRA labels KV1.3 expressing HEK-293 cells.Confocal image of KV1.3 expressing HEK-293 cells labeled with a fluorescent Agitoxin-2-Cys-TAMRA (#RTA-420-T). KV1.3 expressing HEK-293 cells were grown on glass cover-slips, incubated in the presence of 50 nM Agitoxin-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 Rhodamine filter. Right: Merge of both images. As can be seen, Agitoxin-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.
- Alomone Labs Agtitoxin-2-Cys-TAMRA binds to HEK-293 cells stably expressing KV1.3 channels.Top: Staining of HEK-293 cells stably expressing KV1.3 channels with 50 nM of Agitoxin-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 Rhodamine 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 Agitoxin-2 (#STA-420). The same preparation as the one shown on top was pre-incubated for 30 minutes with 10 µM of Agitoxin-2, followed by 30 minute incubation after the addition of 50 nM Agitoxin-2-Cys-TAMRA. Bottom: Agitoxin-2-Cys-TAMRA does not stain non-transfected HEK-293 cells.
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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 Agitoxin-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 (#STI-400) and the KV1 blocker Hongotoxin (#RTH-400).3-4
A similar approach was taken by Alomone Labs in producing Agitoxin-2-Cys-TAMRA. A fluorescent dye is attached to a free Cys residue, which replaces Asp in position 20 of the 38 amino acid long peptide. This novel toxin retains the blocking activity of the original toxin and specifically labels living cells expressing KV1.3 channels.
Agitoxin-2-Cys-TAMRA (#RTA-420-T) is a highly pure, recombinant, and biologically active conjugated peptide toxin.
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