Air dry the tube for 5 to 10 min. Add See Critical Parameters for suggestions regarding controls.
Detect signal by immunoblotting Transfer proteins onto a nitrocellulose membrane e. Wash the membrane three times with PBS-T, 5 min each time. Incubate the membrane with streptavidin-linked horseradish peroxidase in PBS-T for 1 hr at room temperature. Wash the membrane three times, each time with 5 ml PBS-T, 10 min each.
Capture signal on film with 1- to 2-min exposure time. Check for protein incorporation of fatty acid probes via amide or thioester linkages This protocol involves the use of hydroxylamine and is carried out right after protein transfer step 33 above.
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Analyze by streptavidin blot as described above steps 35 to Demonstrate equal levels of protein loading This can be carried out either after step 38 or There are several ways to check for levels of protein loading which include: Coomassie staining total protein levels , anti-actin, anti-tubulin, etc. Below is a procedure for detecting tubulin. Incubate the streptavidin blots with Restore western blot stripping buffer for 15 min at room temperature. Repeat steps 34 to Repeat steps 37 to The membrane was soaked in PBS-T buffer A or in hydroxylamine B , and the signal was detected by streptavidin-linked horseradish peroxidase see Basic Protocol 1.
Alk-C14 yields more labeled bands than Alk-C Scan films using a desktop scanner and import images into ImageJ or AdobePhotoshop.
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Detect signal by in-gel fluorescence In cases where fluorescence scanners are available, use of rhodamine-azide is recommended as it results in minimal background and high signal-to-noise ratio relative to biotin-azide. Follow steps 15 to 32 above, except replace biotin-azide with rhodamine-azide in step Minimize exposure of the gel to light while carrying out the protocol. Export and process the image in the Typhoon scanner image-processing software. It allows the user to capture a view of the spatial localization and dynamics of myristoylated and palmitoylated proteins under different conditions and cellular states.
Perform step 1 in Basic Protocol 1. Prepare the fatty acid probe-containing medium 3. Perform steps 4 and 5 in Basic Protocol 1. Treat the cells with fatty acid probes 4. Aspirate the growth medium from wells containing seeded cells. Wash the cells gently once with 1 ml PBS. Fix and permeabilize the cells 7. Wash the cells four times, each time with 1 ml PBS to remove excess probe. Aspirate the methanol. Wash the cells extensively six times, each time with 1 ml PBS. Label the cells with rhodamine-azide Remove the coverslips from the wells and place them in a humidified chamber for subsequent steps.
Images show the perinuclear and punctuate distribution of cellular proteins labeled with Alk-C14, Alk-C16, and Alk-C Add the click reaction cocktail step 13 above to the coverslips and incubate for 1 hr in the dark at room temperature in the humidified chamber. Mount the coverslips onto the microscope glass slides. Capture images by fluorescence microscopy Acquire 50 to 70 optical Z-sections per image with 0. Analyze the images Export and process the images in a standard image analysis software, such as Slidebook 5.
Biotin-azide or rhodamine-azide Reagents can be obtained from our laboratory, synthesized as described earlier Lewis et al.
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Protect from light. Prepare fresh. Fatty acid stock solution Dissolve lyophilized fatty acid powder [obtained from our laboratory or synthesized as described earlier in Hannoush and Arenas-Ramirez ] in dimethyl sulfoxide DMSO to a concentration of 50 mM. Lysis buffer mM sodium phosphate, pH 7.
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Tris 2-carboxyethyl phosphine hydrochloride TCEP Dissolve powder in water to a concentration of 50 mM just prior to use in the click reaction see Critical Parameters. Radioactivity has been the standard method for detecting myristoylation and palmitoylation of cellular proteins.
Typically, [3 H]- or [ I]-labeled myristic and palmitic acids are added to cells for metabolic incorporation into cellular proteins Schlesinger et al. The signal is developed by autoradiography and requires lengthy film exposures. Because of the hazards and costs associated with radioactivity, this method is less than ideal for use.
Recently, the acyl-biotin exchange method was introduced for detecting protein palmitoylation Drisdel and Green, This method relies on blocking the free sulfhydryl groups of palmitoylated proteins, followed by cleavage of the palmitate moiety and subsequent tagging of the unmasked cysteine with biotin for affinity capture and enrichment. While this method is a breakthrough, it has limitations primarily associated with false positives Roth et al. For a detailed description of the various methods and their limitations, the reader is referred to a comprehensive review on this topic Hannoush and Sun, Recently, nonradioactive probes based on alkyne fatty acids have been developed for metabolic labeling of myristoylated and palmitoylated proteins Hannoush and ArenasRamirez, We reasoned that appending an alkyne group to the terminal end of a fatty acid would not interfere with the hydrophobic nature of the fatty acid and the mechanism by which it inserts into lipid membranes.
The alkynyl fatty acid probes are portable and can be stored indefinitely in the freezer for immediate use. They are metabolically incorporated into fatty-acylated proteins in human and mouse cell lines. The tagged proteins are then conjugated to biotin-azide or rhodamine azide via a click reaction and detected by immunoblotting or in-gel fluorescence, respectively. Additionally, these probes enable cellular imaging of the global subcellular distribution of fatty-acylated proteins and shed light on the dynamics and turnover of such proteins Hannoush and Arenas-Ramirez, Based on hydroxylamine sensitivity, Alk-C14 is a probe for protein myristoylation while Alk-C16 and Alk-C18 are probes for protein palmitoylation.
Other emerging applications of these probes include in vitro labeling of recombinant proteins, monitoring turnover of palmitoylated proteins, enrichment of trace proteins, proteomics studies, and immunoprecipitation of specific proteins of interest for profiling their fatty acylation status Heal et al. The toxicity of the copper reagent makes it inappropriate for monitoring fatty acylation by live-cell imaging. Furthermore, the presence of certain endogenous proteins that chelate copper may interfere with the signal of the assay, and this varies depending on the particular cellular system being studied.
Critical Parameters Reagent preparation and concentrations Avoid multiple freeze-thaw cycles of the fatty acid probe stocks as this may affect reagent quality. Also, copper sulfate and TCEP reagents should be freshly prepared just prior to use in each experiment. Experimental controls Two protein gels should be run simultaneously for each set of samples. One gel is run and analyzed as described in steps 31 to 38 Basic Protocol 1 , while the other gel is treated with hydroxylamine and analyzed as described in steps 39 to 41 Basic Protocol 1.
This allows the user to determine the type of linkage via which fatty acid probes incorporate into cellular proteins. Sometimes, detection by streptavidin-HRP results in varying degrees of background staining, which stems from streptavidin labeling of endogenously biotinylated proteins. Therefore, care should be taken to decipher nonspecifically labeled bands on the gel, and it is critical that the researcher include negative controls, such as those lacking copper sulfate or the fatty acid probes. Furthermore, for imaging by fluorescence microscopy, it is critical to ensure that the signal observed is not due to nonspecific incorporation of the fatty acid probe into biological membranes.
Therefore, optimizing the time for methanol fixation and the permeabilization step for the particular cell type used is crucial to avoid nonspecific incorporation into lipid bilayers. The intensity of staining will decrease with longer detergent extraction times if there is nonspecific incorporation of the probes. Troubleshooting While the protocols described herein detail a robust method for detecting protein myristoylation and palmitoylation, in very few cases a signal may not be observed and below are recommended troubleshooting procedures.
Reagent quality and concentration Fatty acid probes were not used at the appropriate concentrations to obtain the best signal-to-noise ratio. Also, the click chemistry reaction works best only when the reagents are freshly prepared just prior to the experiment. Signal optimization Sometimes, staining is not homogeneous throughout the membrane blot.
This may be due to uneven transfer of proteins from the gel. In this case, ensuring that the appropriate transfer procedure is used is critical. Also, if the intensity of bands on the membrane is high, then reducing exposure times or using a more diluted stock of streptavidin-HRP can resolve this issue. Another source of reduction or loss in signal is proteins that may interfere with the click reaction, such as those that may chelate copper.
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