To test the interaction between two proteins, the two gene of interest need to be cloned into our BF and vectors using gateway longing technique. This constructs are transformed into aerium GV 3 1 0 1. The GV 3 1 0 1 bacterial cultures containing to construct will be mixed and co infiltrated into the leaves.
If the two candidate proteins interact with each other, the YFP signal can be observed using outcome focal Mexico. The main purpose of BFC technique is to test interaction between two proteins. In vavo, we have created a series of gateway compatible BFC and IS two hybrid vectors which provide researchers with easy to use tools to perform both BFC and ISTO two hybrid assays.
In this video, we are going to demonstrate the ease of using our BFC system. To test the protein, protein interactions in unbe manner transcend Expression system. First you need to Prepare the GV 3 1 0 1 bacterial culture.
You need to pick a single colony from your plate and inoculate in five male of YEB medium with Appropriate antibiotics. Then you need to shake This cultures overnight at 28 degrees. The speed is 200 runs per minute.
The bacterial cultures should be ready the next morning. Make sure the bacteria D over grew. Take the cultures out from the incubator and transfer one male of each culture into a sterile 1.5 male Centrifuge tube.
Put the centrifuge Tubes into a desktop centrifuge. Spin down the bacteria at 1000 G for 10 Minutes at room temperature, discard the snet. Then watch the penate by adding one meal of infiltration medium and the reassessment by per Petting a few times.
Repeat the wash step For two more times. This is important because it helps to remove the trace of antibiotics in the bacterial medium, which make you plant seals after infiltration. After the final wash, resus suspend the pallet in 0.5 male infiltration medium measures owed 600 of this bacteria suspensions using a spectro photometer using infiltration medium as bank control.
After the OD measurement, you need to adjust the OD using infiltration medium. The final OD should be between one and 1.2. You should prepare all the bacterial suspensions before proceeding to the next step.
Transfer equal amount of bacterial suspensions corresponding to your two candidate proteins and mix them in a new 1.5 male tube Negative bacteria mixture is now related to infiltrate. Prepare all the combinations and don't forget to Include a negative control. You should use five to six Week code and best manner plants for infiltration.
You can remove the plants from the growth room and place them on We night for one hour before infiltration to open the storm attack. This will help with the infiltration process. Draw up about 100 microliter of Resus suspended bacteria.
Mix near one male slipped tip syringe without the needle. Place the tip of the syringe against the underside of the leaf ly. Support the upper side of the knee with your finger.
Generally press down on the plunger. You should be able to see the liquid diffusing through the leaf as yet feels the Meso thinner Air Spaces After Infiltration enable the infiltrated aero with a mark pen. Otherwise, after two days, you will not be able to identify the infiltrated A.If you need to infiltrate With different bacterial mix mix, make sure to spray your glove with 50%ethanol or change your G gloves between Infiltrations.
Try to leave a One meter rib space between infiltrations to prevent cross-contamination. The PFA signals should be monitored Every 24 hours from infiltration up to five days. You can use two cover sleeves of different Sizes to hold the sample.
First, put one drop of mini water on a notch cover sleeve. Then cut a small piece of knife tissue within the infiltrated zone. Try to avoid nausea Vein in the sample.
Put the cut knife tissue in the water on the slide Under set down, cover it using a smaller current sleeve and depress gently. Now the sample can be observed under the Mexico scope. Here we use this NACA T CSS P two confocal Mexico to visualize the BIFC signal.
Even regular scent Mexico can be used. Confocal provides better pictures and detect weak signals. You can use the YFP parameter to detect the VFC signals in this NCAs P two system.
You can simply double click on this YFP preset here, which will automatically turn on the excitation and set the emission detection range. If you are using a different system, please refer to the manual to properly set the parameters. It is our hope that after watching this video, you should be able to conduct the BFCE to test protein protein interactions using unbe MI Transcend expression system.
You can also apply this procedure to tobacco and AY plants. Our BFC vectors also contain an HA and flag tag respectively, so you can also perform co IP to validate the interactions if necessary.
