Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

The overall goal of the following experiment is to demonstrate a protein receptor interaction using surface plasma and resonance. This is achieved by injection of the nickel ions over the nitro tri acetic acid or NT a matrix. Next one of the proteins termed the ligand is immobilized onto the sensor chip following a blank injection.

The second protein termed the analyte is injected over the immobilized ligand. In order to detect an interaction between the two proteins, the results show that the two proteins interact transiently the analyte associates, and dissociates from the ligand. The affinity of the interaction can be calculated based on the measured dissociation and dissociation rates of the two proteins.

The main advantage of this technique over existing methods like pull down or immunoprecipitation, is that it can identify transient or low affinity interaction that are often of great biological consequences. This method can help answer key questions In biochemistry such as whether the protein of interest binds to another protein or another small molecule, the nature of the interaction can be determined and the affinity can be measured. To prepare the sample, remove all aggregates from the purified protein as described in the text protocol.

Prepare the running buffer and filter all buffers and protein samples using zero point 22 micron filters. Ensure that the filters are protein compatible in advance before connecting the running buffer bottle to the pump inlet. Keep aside 10 milliliters of the running buffer in a separate tube.

Then dilute the samples against the running buffer to avoid buffer mismatches. As a rule of thumb, for stable ligand demobilization use low ligand concentrations with longer injections at low flow as opposed to high ligand concentrations With shorter injections at high flow Dilute the concentrated ligand stock into running buffer to a final concentration of approximately 20 micrograms per milliliter. Finally dilute the analyte into the running buffer to the desired concentration.

For a system of unknown affinity, test a range of analyte concentrations from 10 micromolar to 10 ano molar in tenfold dilutions. In this demonstration, an NTA coupled chip suitable for capturing proteins with an oli histidine tag is reused. After taking the stored chip from the buffer, rinse thoroughly with double distilled water, dry gently, and place into its original sheath.

To dock the chip onto the SPR instrument, open the sensor chip door and place the chip in its sheath. Close the door and press dock chip. Set the chip cell temperature to 25 degrees Celsius.

Set the samples compartment to seven degrees Celsius to keep the protein stable throughout the experiment. After preparing the solutions for loading and stripping as described in the text protocol, prime the SPR instrument with running buffer. For the next step, set the flow to 50 microliters per minute.

Define the flow paths and reference subtraction to obtain the results described here. Set the paths to flow cell number one and flow cell number two and flow cell number one, subtracted from flow cell Number two, after selecting the suitable sample rack, save the results file before starting the experiment so that the data will be saved during the run. Each experiment is composed of cycles.

Keep a clear record of the injections done in each cycle and separate the ligands loading the buffer's blank injection and the analyte injection into different cycles. Cycle one is chip preparation and nickel loading. Make sure the flow and paths are set and then inject 350 millimolar EDTA for one minute to wash away remaining molecules.

Next, set the flow to 10 microliters per minute. Then inject 0.5 millimolar nickel chloride for two minutes. Now the chip resin is coated by nickel ions.

Cycle two is ligand immobilization. Set the flow to 15 microliters per minute and set the flow path to flow cell. Number two, inject ligand A until reaching response unit or R ru values that are 10 to 20 fold of the ligand molecular weight.

For example, load a ligand of 50 kilodaltons to between 501, 000 rus. Keep a record of the RU value achieved. Then set the flow to 15 microliters per minute and the flow path to flow cell number one, inject control ligand B up to the same RU value as that of ligand a monitor the subtraction, sensori Graham online to help control the injection length.

Next, set the flow to 50 microliters per minute and the flow path to flow cell number one and flow cell. Number two, proceed to wash the system for five to 20 minutes until the baseline stabilizes. Cycle three is the blank injection.

This injection will serve for blank subtraction. Therefore include all components that will be injected with the analyte except the analyte itself. Injection length can vary depending on the time it takes to reach equilibrium.

Set the flow to 15 microliters per minute and set the flow path to flow cell number one and flow cell number two, and flow cell number one, subtracted from flow cell. Number two, insert the weight command for 30 seconds to achieve a stable baseline. Then inject the running buffer for 13 seconds.

Wait 120 to 600 seconds to record the dissociation phase. The length of this step varies according to the kd. The slower the dissociation, the longer this step needs to be.

Cycle four is analyte injection. Copy and paste the exact conditions used in cycle three so that these two injections can be later subtracted. Change the location of the slot in the rack from the one holding the control solution to one that holds the analyte solution.

Choose a concentration that is slightly above the expected equilibrium dissociation constant. If no prior knowledge is available, choose one micromolar as a good starting point. After performing the buffer injection and analyte injection a second time, perform cycle seven chips trip off.

Set the flow to 50 microliters per minute, then inject 350 milli molar EDTA for one minute. Repeat this step two more times. Do not use a single injection of three minutes as this may damage the chip.

Next, inject 0.25%sodium dcal sulfate for one minute. Repeat this step two times. Do not use a single injection of three minutes as this may damage the chip following nickel loading, the transporter of interest is immobilized onto flow cell number two up to 3, 500 R rus.

Then using the same flow and injection duration, the control ligand is injected onto flow cell number one, initially reaching only up to 3000 R to make sure that similar protein amounts are immobilized on both flow cells. The control ligand is further loaded with shorter injection while monitoring the loading up to 3, 500 RDUs. The stairs shape represents the gradual increase in mass on flow cell number one upon each injection shown here is flow cell number one, subtracted from flow cell.

Number two, it is important to vary the duration of the ligands injection to achieve equal loading. In contrast, the duration of the analyte injection must be kept constant for all concentrations used to gain quantitative data. A range of analyte concentrations is injected.

Shown here are injections of different analyte concentrations with a constant injection length. The different analyte concentrations are injected in random order and the double blank sensori grams are x axis aligned prior to analysis to derive the affinity and rate constant. A model fitting is applied.

The calculated equilibrium dissociation constant for the mod BCA interaction is approximately three times 10 to the minus six molar with a moderately fast KA and fast KD Once mastered. This technique can be Done within a few hours after watching this video. You should have a good understanding of how to design and perform an SPR experiment using your protein of interest.