Hello,
As a newbie to SPR, I've been having problems with optimizing aptamer-protein binding on an NTA chip. My protein has a his-tag on it. Any help would be greatly appreciated.
Question 1:
Do the running buffer, analyte buffer and ligand buffer all need to match? ie. The running buffer in the start-up cycle is the same as the buffer to dilute the aptamer analyte and the protein ligand to appropriate concentrations.
Question 2:
My running buffer is: 25 mM Tris-HCl pH7.5, 100 mM NaCl, 20 mM imidazole, 0.005% Tween 20 and 0.5 mg/mL BSA. When I run a startup cycle, the injection of running buffer results in a noticeable increase in RU.
I have also tried: 10 mM HEPES, 150 mM NaCl, 50 µM EDTA. 0.005% Tween 20, pH 7.4. With same results. I read that running buffer should not increase the RU, how can I solve this issue?
Question 3:
As a result when I inject analyte (diluted in running buffer) it also results in an increase in RU. But because I've matched running buffer, analyte and ligand, the resulting Fc=2-1 is flat, with small short dip and peak at injection times. Is this an accepted result?
Question 1:
Do the running buffer, analyte buffer and ligand buffer all need to match?
Yes perfect match to avoid the bulk effect. The best way is to equilibrate your sensorchip in 20 min of running buffer. Then, you can start a start up experiment and the analyte buffer will be your running buffer. For your ligand buffer, it will be the running buffer.
Question 2:
The injection of solution on FC1 and FC2 give you a signal. Normal. You have to check the subtraction of FC2-FC1 to see if the signal is close to 0.
Question 3:
The aptamer has to be diluted in the running buffer. After subtraction FC2-FC1, if you don't see increasing signal, it means that there is no binding. However, aptamers are difficult to work with using SPR. They are negatively charged and the Carboxydextran CM sensorchips are also negatively charged. So, you could have an electrostatic repulsion while you are injecting your analyte in solution. I will recommend you to capture the aptamer with a biotin. And then, inject your protein.
Vanessa
I agree with Vanessa Porkolab's suggestion about capturing aptamer and using protein ligand as analyte. Just one thing I would like to add that when using your protein as analyte, it will be better if you dialyse it in running buffer instead of just diluting, to avoid buffer mismatch.
Yes, the running buffer and the buffer containing the analyte needs to match. It is useful to have a concentrated stock of your analyte to dilute down with running buffer in the hope that it will ultimately be in the running buffer and not the original buffer. You could dialysed your analyte with the running buffer - although, this will not save you time but if it helps then why not. Always do a buffer (blank) run which after a kinetics run you will want to do a buffer subtraction in addition to reference sensor surface subtraction.
If you immobilised your His-tagged protein on a Ni2+ NTA chip followed by amine coupling to ensure that most of your ligand is in a similar orientation followed capping the sensor surface with ethanolamine to prevent nonspecific binding, then you might be okay...With aptamers I guess adding divalent metal ions are needed to keep them stable.
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