I'm working with an his-protein and I'm trying to obtain an IC50 between the protein and the biotinylated substrate and a known competitor. But the experiment is not working. I will add the protocol I'm following :
4 uL HIS-protein = stock 5x ( 500 nM) in 50 mM HEPES, 50 mM NaCL pH = 7.5
4 uL Peptide-Biotin = stock 5x ( 1uM) in 50 mM HEPES, 50 mM NaCL pH = 7.5
5 uL Streptavid-eu criptate = stock 4x ( 8nM) in 50 mM HEPES, 50 mM NaCL pH = 7.5
5 ul Anti-His AB = stock 4x (40 nM) in 50 mM HEPES, 50 mM NaCL pH = 7.5
1 uL compound = stock 20x in 250 mM HEPES, 250 mM NaCl 20 % DMSO pH 7.5
1 uL BUFFER 4 M KF, 10mM CHAPS, 1 % BSA pH 7.5
FINAL conc: 100 nM PROTEIN, 200 nM peptide, 2nM Streptavid-eu criptate, 10 nM Anti-His AB, from 1000 nM to 0.1nm compound with 2 fold dilution ( Kd=10 nM), 50 mM HEPES, 50 mM NaCl 200 mM KF, 0.5 mM CHAPS, 0.05 % BSA 1% DMSO pH 7.5
Procedure :
Thermo Scientific™ Nunc™ 384-Well Optical Bottom Plates (Thermo Scientific™ 242764)
The experiment seems not working since no competitions is appearing.
QUESTIONS :
1) How do you mix the components of this BUFFER 4 M KF, 10mM CHAPS, 1 % BSA pH 7.5 because I'm having problems, when mixing KF with CHAPS it appears a white colloidal solution that is imposible to solubilize.
2) Which is the Fluorescence response that i should expect for donor and acceptor? I usually have a fluorescence intensity around 20000 for the donor and 1000 for the acceptor. I always calculate the 665/620 ration *10000 and it gives me from 100 to 200.
3)Would it be better to use white plates, low volume like CORNING 4513?
4)Should I add CHAPS in the protein buffer instead of adding it at the end?
6)Should I change some parameters for the reading in the clariostar?
My experience to Q2-Q6:
2) If a fluorescence intensity for 620 is 120000 for example in a negative ctrl, a intensity for 665 may be 100000 in positive ctrl and the corresponding one for 620 is 20000. I attach a csv file as a detailed example.
3) HTRF should always be peformed in opaque bottom plates, white bottom in most cases for a higher signal. We use ProxiPlate in 384 format from PerkinElmer for most HTRF assay.
4) I suggest you refer to manufacturer's instructions. In our lab's protocol, assay buffer is refer to the environment for kinase reaction, in which the protein you are interested in and biotinylated peptide are diluted. The procedure is: adding 2 μL compounds in 10% DMSO→adding 4 μL protein (optional: incubate depending on different assay mechanism) in assay buffer→adding 4 μL biotinylated peptide in assay buffer and incubate for 1h→adding 5 μL stop buffer→adding 5 μL detection solutin containing xl665 and europium cryptate labeled streptavidin in detetion buffer→incubate in dark place and read plate.
6) If the microplate reader offered a "standard" reading protocol, you should optimize the parameters in the end. As you are using kits from cisbio, please refer to application notes, guideline or technical notes from cisbio (https://www.cisbio.cn/resources or in the product page of your kits). If the ratio or signal is still unacceptable, here is a example in the attech file.
Hello,
I did a matrix of several concentrations to maximize signal response, e.g. successively increasing the acceptor probe and keep the other components constant. Repeat the same approach for each component until you see higher FRET efficiency. It worked with my biotin-RON2 and histag-AMA1 malaria complex
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