The overall goal of this procedure is to detect low abundance growth factor receptors in zebrafish embryos by combining the selectivity and sensitivity of affinity labeling of the chosen receptor by its radioactive ligand. This method can help answer questions in developmental biology fields such as receptor expression levels and their biochemical properties. This method which we have nicknamed AFLIP extend a powerful descriptive qualities of affinity labeling, a well-established cell biology technique to zebrafish developmental studies.
To begin this procedure, collect 100 to 200 embryos for each condition and place them in petri dishes containing fish water. Then manually dechorionate the embryos using fine-tipped forceps and avoid using pronase or any other protease throughout the protocol as it may digest the targeted receptors. Next, place the embryos in a 1.5-milliliter microfuge tube and wash them twice with 1X PBS.
Then add 500 microliters of deyolking buffer. Release most of the yolk by gently pipetting the embryos in the solution up and down about 30 to 40 times. For 72 HPF and 48 HPF embryos, use blue and yellow pipette tips respectively.
Yellow tips may need to be slightly cut to avoid destroying the embryos. A successful yolk release can be checked by observing the embryos under the microscope. Removal of yolk must be as complete as possible as yolk lipids and proteins may interfere with subsequent ligand or antibody binding steps.
After that, collect the embryos by centrifugation at 600 x g for 15 seconds. Carefully discard the supernatant using a pipette. Next, wash the embryos twice with 500 microliters of washing buffer by gently vortexing them at the lowest speed.
Then collect the embryos by centrifugation at 600 x g for 15 seconds. From here onwards, continue the procedure at four degrees Celsius. Discard the supernatant carefully using a pipette.
Afterward, we suspend the embryos in 350 microliters of lysis buffer and homogenize them using a plastic pestle. Then incubate the lysed embryos for 30 minutes with agitation on a test tube rocker at four degrees Celsius. Next, centrifuge the lysed embryos at 11, 000 x g for 15 minutes in order to remove the insoluble debris.
Following that, transfer the cleared supernatant to a fresh tube. Determine the total protein by the Bradford protein assay and plotty calibration curve. In this step, place 400 to 500 micrograms of total embryo protein in a 1.5 milliliter microfuge tube and dilute it to one microgram per microliter with buffer 1.
Next, add Iodine-125 label TGF-beta II stock to reach a final concentration of 150 picomolar and incubate with agitation on a test tube rocker for two hours at four degrees Celsius. After that, add undiluted antibody against the receptor of interest in order to reach a 1:100 dilution. Then continue the incubation at four degrees Celsius for another two hours or overnight.
After incubation, add 50 microliters of suitable immunoglobulin binding beads and incubate for 50 minutes with agitation on a test tube rocker at four degrees Celsius. Then recover the beads by microcentrifugation at 11, 000 x g for 20 seconds and discard the supernatant in an appropriate radioactive trash container. Subsequently, wash the IP beads three times with one milliliter of IP wash buffer by vortexing at microcentrifugation at 11, 000 x g for 20 seconds each time.
Then resuspend the IP beads in 250 microliters of IP wash buffer. Prepare fresh DSS solution and add 1.5 microliters of it to the sample. Following this, incubate the sample for 15 minutes at four degrees Celsius with agitation.
In order to quench the cross-linking reaction, add 500 microliters of IP wash buffer supplemented with enough tryst chloride stock at pH 7.4 to reach 25 millimolar tryst chloride. Next, centrifuge at 11, 00 x g for 20 seconds to collect IP beads. Afterward, discard the supernatant and resuspend the IP beads in 30 microliters of reducing laemmli buffer.
Next, boil the sample at 94 degrees Celsius for five minutes before SDS-PAGE gel electrophoresis and then analyze the dried gels in a phosphorimeter or by conventional autoradiography. This figure shows the representative results obtained with AFLIP. Total protein extracted from the embryos at 72 hpf and 48 hpf were subjected to Iodine-125 TGF-beta II affinity labeling followed by IP with rabbit anti-zebrafish BG or rabbit anti-rat BG as a control.
In the autoradiography, BG can be detected without GAG or as a glycosaminoglycan-modified BG.This figure shows how AFLIP can be used to gauge the expression levels of a given receptor in embryos subjected to an experimental manipulation, in this case, the decrease of betaglycan to the morpholino injection. Lane one shows the level of TGF beta receptor III in a 72 hpf wild-type embryo, while lane two shows the effect of administering seven nanograms of a morpholino directed to the exon 2-intron 2 boundary of the ZFPG primary transcript. Note that the BG downregulation obtained with this morpholino is not reproduced by its mismatched control.
These results confirmed that the phenotype obtained with this morpholino was specific to knockdown of the TGF beta receptor III as described before. The success of AFLIP for detecting a growth factor or cytokine receptors depends on the availability of a radiolabeled ligand that can be bound and cross-linked to its receptor. A good antibody for immunoprecipitation is also require.
Once mastered, this technique can be done in two days if it is performed properly. Since AFLIP avoids the transfer step of the western blot, it allows for an enhanced detection of heavily glycosylated receptors. And we have seen here for the proteoglycan betaglycan which transfer very inefficiently in western blot Following this procedure, other methods like enzymatic digestion can be performed in order to describe additional post-translational modification as long as they are revealed by electrophoretic techniques.
After watching this video, you should have a good understanding of how AFLIP can help you detect low abundant receptors in several fish embryos by affinity labeling linked to immunoprecipitation. Finally, don't forget that working with Iodine-125 must be done with care and adequate protection. Good luck with your experiments.
