Q&A

How much and what type of material do we need to prepare as a protein source? Extracts from cultured cells and organs, as well as plasma proteins, are applicable. when using cultured cells as a protein source, up to 109 cells for nuclear proteins and between 107 and 109 cells for cytoplasmic proteins might be needed.

Can we “Fish”for GPCRs or Ion channels as targets? It is not impossible, but probably difficult to fish for GPCRs or Ion Channels using our method. Membrane proteins can be fished if they are solubilized by detergent.

What is the binding affinity of the fished protein? The binding affinity of fished proteins to compounds is usually below 10-6 M Kd. In cases where the binding affinity is low (10-6 - 10-7), the background noise will be high.

How long is the shelf life of FG-beads? FG beads have a shelf life of 12 months at +4℃ after the date of manufacture. The beads should not be frozen.

How can we select the type of linker? There are several types of FG beads available bearing linker termini with different functional groups. FG beads should be selected based upon the functional group of the chemical probe.

What key poins shuld we consider or pay attention to before designing a chemical probe? When you design a chemical probe for a certain target protein, you should consider introducing additional functional groups in the probe. However, it is often observed that the same probe binds different proteins, as a result of the steric effects caused by the position of the linker connected to the beads. Therefore, if possible, we recommended introduceing a probe to linkers at several positions and running tests to fish the desired target proteins. If there are no active functional groups in the chemical structure of the probe, a special probe should be synthesized to obtain the appropriate chemical structure.

Required amount of beads For preliminary experiments consisting of two Receptor Fishing tests, 20 mg of beads shuould be sufficient. In order to obtain a substantial amount of purified target proteins, 40-50 mg of beads will be required following the preliminary experiments.

Tethering process (beads / chemical probe) The binding yield for the functional group of the bead linker terminal depends upon the functional group of the chemical probe. NHS beads can readily react with a chemical probe bearing a NH2 functional group.

NHS ester content per beads’weight is approximately 200 nmol/mg. Coupling reactions are conducted with three concentrations of probe solution: 0.1 mM, 0.3 mM, and 1 mM) The contents of the combined chemical probes depend on the nature of each compound. The range of contents is from a few nmol to 100 nmol. Good results are generally obtained under the mid-range conditions (a few nmol up to 20-30 nmol). However, an excess of combined contents causes an unacceptable steric disadvantage for fishing.

Introduction of beads to a chemical probe If necessary, we can provide such services and will send a quote for your specific research needs. We also offer probe synthesis services. Please contact us.

Magnet Device Our recommend Magnet for FG beads is "DynaMagTM-2." Please see the URL below:
DynaMagTM-2.

Success rates of fishing binding protein & Identification The success rate is almost 50% in our in-house research, when we use a chemical probe that binds to the target proteins with high affinity. However, the success rate will depend on the nature of each compound.

Payment method We do not accept personal checks, but we accept money orders in Japanese yen. If you prefer payment by money order, bank transfer, or credit card, please contact us, and we will promptly provide payment instructions.

Courier selection and shipping fees We guarantee the quality of FG beads under cool delivery　cinditions. Due to international cool delivery requirements, we usually use World Courier delivery and the shipping fees are not inexpensive. If the customer requests, we can use other available carriers to make shipments at room temperature; however, in such cases, we cannot guarantee the quality of the FG beads.
If the customer has an account with World Courier and/or another cool delivery carrier, we can arrange cash on delivery without a problem. If you desire such special arrangements, please contact us.