In the IP FCM experiments, protein complexes are captured on beads in order to identify and study the different proteins interacting together. First, an immuno precipitating antibody specific for protein of interest is covalently coupled to polystyrene latex beads. As a second step, these beads are incubated with cell lysates, allowing the specific protein and interacting proteins to bind to the beads.
The beads together with the captured proteins, and then washed and probed with fluorescently labeled antibodies specific for various molecules that may be associated with the immuno precipitated protein. Finally, the fluorescently labeled beads are analyzed by flow cytometry to determine which proteins are present on which you're absent from the captured complexes. The advantages of this technique over other methods, such as immunoprecipitation, followed by western blotting, is the ability of I-P-F-C-M to allow assessment of native protein protein interactions without requiring genetic engineering or large sample sizes.
I-P-F-C-M can yield a high signal to noise ratio even when very little biomaterial is available for analysis as it can be performed with small volumes and low concentrations of analytes. To begin this procedure, prepare the following buffers, MES coupling, buffer quenching blocking storage buffer, abbreviated QBS and flow cytometry or FCM buffer. Determine the concentration of the carboxylate modified polystyrene latex beads or CML beads by diluting in PBS and counting with a hemo cytometer.
Once the concentration of the beads is determined, pipette 18 times 10 to the six beads into a 1.5 milliliter micro centrifuge tube. Wash the beads two to three times in 0.5 to one milliliter of MES coupling buffer centrifuging at 20, 000 times gravity for three minutes at room temperature. After each wash while the beads are centrifuging.
Prepare fresh e dac MES buffer for use in activating the beads. Once the washes are complete, keep the buffer either at room temperature or on ice. After the final wash, resuspend the beads in 50 microliters of the MES coupling buffer.
Then add 20 microliters of EEC MES to the bead suspension. In order to activate the carboxyl groups on the beads, mix gently at room temperature by manually pipetting up and down continuously for 15 minutes. Although this step is labor intensive, this method of mixing gives the best results After mixing.
Wash the activated beads two to three times in 0.5 to one milliliter of PBS Centrifusion at 20, 000 times. Gravity for three minutes at room temperature After each wash after the final wash, resuspend the activated beads in 50 microliters of PBS to couple the IP antibody to the beads. Add 50 microliters of the IP monoclonal antibody from a stock concentration between 0.2 to one milligram per milliliter.
Make sure that no primary amine containing molecules other than the antibody are present. These amine containing molecules such as tris or bovine serum albumin compete with the antibody for coupling to the beads. Mix the bead antibody suspension on a vibrating shaker for three to four hours at room temperature.
Shake sufficiently to prevent settling of the beads on the bottom of the tube. After binding. Wash the antibody cod beads two to three times in 0.5 to one milliliter of PBS centrifuging at 20, 000 times.
Gravity for three minutes at room temperature after each wash. Finally resuspend the beads in 100 microliters of QBS buffer. Coupling of monoclonal antibodies to CML beads can be checked by staining the beads with fluro conjugated antibodies against IgG.
Followed by FCM analysis, the beads coated with the monoclonal antibody can be stored at four degrees Celsius until ready to use in an IP experiment. First, suspend the beads well dilute one to 200 to one to 10, 000 in PBS and count with a hemo cytometer. The concentration must be measured for each preparative batch so that the number of beads used in each IP experiment can be precisely controlled.
Next, prepare fresh dig in solution in water at 2%weight per volume. Heat to 95 degrees Celsius for five minutes. Then cool on ice.
Dilute, dig into 1%weight per volume in freshly prepared lysis, buffer, and keep on ice. Determine the number of cells to lys using the table shown as a guideline. However, the number of cells required depends on the protein of interest and must be determined empirically.
Here, 30 times 10 to the six cells from a small lymphocytic tumor cell line are used. Transfer the required cell suspension volume to a 1.5 milliliter centrifuge tube. Pellet the cells at 20, 000 times.
Gravity for 30 seconds at four degrees Celsius and aspirate the media. Add the appropriate amount of freshly prepared. Dig lysis buffer to the pellet mixed gently by pipetting up and down.
Then allow the cells to lice for 20 minutes on ice. Note that depending on the cell type and the protein protein interactions studied cell lysis conditions can vary and must be determined empirically when lysis is complete. Remove the nuclei and insoluble cellular debris by centrifuging the lysate at 20, 000 times.
Gravity, the 10 minutes at four degrees Celsius. Keep the SNA and discard the pellet to bind the target protein to the antibody coated beads. Add 50, 000 to 250, 000 monoclonal antibody coupled beads to the clarified lysate pipetting gently.
To mix the minimum volume of lysate used for a single IP is five microliters. Place the clarified lysate and bead mix on a rotating vertical wheel for four hours to overnight in a cold room set to a sufficient velocity to prevent the beads from settling below volume ips, the liquid can remain at the bottom of the tube during rotation. Following the binding of the target protein to the IP antibody.
Proceed to fluorescently label the immuno complex. Wash the IP beads two times in one milliliter of ice called FCM buffer, centrifuging at 20, 000 times gravity for three minutes at four degrees Celsius. After each wash, re suspend the beads in 100 microliters of FCM buffer and aliquot 20 microliters into each of five or more.
1.5 milliliter micro centrifuge tubes or wells of a chimney bottom plate. Both are shown here. Add fluorochrome conjugated monoclonal antibodies to the samples.
Perform the FCM stain following the vendor's instructions or according to empirically determined concentrations as a starting point. Add 0.2 to one microliter of stock antibody solution of at least 0.1 milligrams per milliliter per tube or well and incubate for 40 minutes on ice. Wash probed beads in 1.5 milliliter tubes two times with one milliliter of ice cold.
FCM buffer, centrifuging at 20, 000 times. Gravity at four degrees Celsius for three minutes. After each wash, gently remove the supinate after each wash, taking care not to disturb the beads.
Alternatively, for a chimney, bottom plate wash two times with 0.2 milliliters of ice cold, FCM buffer centrifuging at 1000 times gravity for five minutes at four degrees Celsius. After each wash, after each wash. To remove the supernatant from the beads, flick the plate once then while still upside down blot any drips from the edges of the wells.
The residual volume in each well containing the beads will be about 20 microliters. Suspend the beads in 200 microliters of FCM buffer per sample transfer to labeled fax tubes. The samples are now ready for flow cytometry or FCM on Becton Dickens and flow cytometers.
Remove the droplet containment sleeve that may surround the sample inlet or sip. Allow the sip to drip between samples as this will prevent beads from being carried over from one sample to another. The CML beads used here are three to five micrometers in diameter, approximately half the diameter of a quiescent mouse lymphocyte.
Therefore, it may be necessary to manually increase the forward scatter amp gain and the side scatter voltage in order for the population of bead events. To register on the cytometer here, this is done using unlabeled or blank beads. The settings in gait are adjusted to exclude bead, doublets and abri prior to reading.
Experimental samples ensure that the fluorescence will be on scale. The unlabeled beads serve as the negative control of fluorescence and rainbow calibration particles or RCPs can be used as a positive control of fluorescence. Since RCPs are smaller than the CML beads, forward scatter and side scatter parameters as well as gait position may need to be temporarily altered for that sample.
After these calibrations, the negative and positive fluorescence extremes are visualized on a log axis. Once calibration with RCPs is complete return to unlabeled bead settings for forward and side scatter. As long as only one fluorescent probe per sample is used, as in the experiment shown here, no fluorescence compensation is necessary.
Now acquire and save the fluorescence data files analysis is performed subsequently using flow cytometry software such as cell Quest or FlowJo. Here is the result of an IP FCM analysis of mouse T-cell LY in 1%digit toin. The TCR CD three non-protein complex was pulled down with anti CD three epsilon beads.
The captured complexes contains significant quantities of TCR beta and co associated CD three epsilon. Close to background levels of other proteins were detected as determined by using an irrelevant immunoglobulin probe or antibodies against the proteins TH 1.2, CD 45, or H two KD.The advantages of this technique over other methods such as immunoprecipitation, followed by western blotting, is the ability of I-P-F-C-M to allow assessment of native protein protein interactions without the need for genetic engineering or large sample sizes. I-P-F-C-M can yield high signal to noise ratios even when very little biomaterial is available for analysis as it can be performed with small volumes and low concentrations of analytes.
