This concanavalin A-based in vitro sedimentation assay is designed to measure binding affinity of glucan phosphatases against different STA substrates. The information we gain from this method is fundamental to our understanding of glucan phosphatase family of enzymes. Carbohydrate protein interactions are relatively weak, therefore current methods cannot detect glucan phosphatase and glucan interactions.
Our lectin based cosedimentation assay can detect binding affinity in the millimolar range. The characterization of SEX4 mutants as well as other members of the glucan phosphatase family of enzymes demonstrates the ability of this assay to quantify protein carbohydrate interactions. One of the most challenging parts of the assay is to optimize the concentration range of the substrate.
We recommend starting with a broader range to get an idea and then narrowing down for a specific range. To begin, pipette 250 microliters of ConA-sepharose beads suspension into a 1.5 milliliter microcentrifuge tube. Centrifuge the contents at 10, 000 G for 30 seconds at four degrees Celsius.
After discarding the supernatant, add 750 microliters of the binding buffer to each tube containing 250 microliters of ConA-sepharose beads. Centrifuge the tubes at 10, 000 G for one minute at four degrees Celsius and remove the supernatant. Next, prepare twofold dilutions of the solubilized amylopectin solution to make a series of two milliliters diluted amylopectin solutions.
To prepare the ConA-sepharose amylopectin beads, add 250 microliters of each diluted amylopectin solution to the tubes containing 250 microliters of ConA-sepharose beads preequilibrated and binding buffer. Mix the contents well and label the tubes with the corresponding amylopectin concentration. Incubate the tube contents on a rotating wheel at four degrees Celsius for 30 minutes and centrifuge the tubes at 10, 000 G for one minute.
Mix 250 microliters of ConA-sepharose amylopectin beads with 100 microliters of the binding buffer containing 10 micrograms of starch excess four, or SEX4 protein, 10 millimolar of dithiothreitol, and 10 micromolar of protease inhibitor cocktail. Incubate the contents at four degrees Celsius for 45 minutes with gentle rotation. Then centrifuge the tubes at 10, 000 G for one minute and carefully pipette 50 microliters of the supernatant into a new 1.5 milliliter microcentrifuge tube.
Add 20 microliters of 4X SDS page dye and 10 microliters of distilled water to each tube containing 50 microliters of the collected supernatant fractions and label them as S.Similarly, add 20 microliters of 4X SDS page dye and 80 microliters of distilled water into the tubes containing washed ConA-sepharose amylopectin SEX4 beads. After adding dye and water, heat all the samples at 95 degrees Celsius for 10 minutes. Once the ConA-sepharose amylopectin SEX4 beads are centrifuged at 10, 000 G for one minute, save the supernatant and label it as P.Load 40 microliters of the unbound protein samples labeled as S into 4 to 12%precast polyacrylamide gel wells from the lowest substrate concentration to the highest, reserving the first lane for a protein molecular weight marker.
Use a second gel to load the glucan bound protein samples labeled P.Add freshly prepared SDS page running buffer to both chambers of the apparatus and run the gel at 150 volts for 35 minutes or until the dye front reaches the bottom of the gel. Then take the gel cassette from the apparatus and remove the spacers and glass plates to separate the gel for western blot analysis. Make one liter of transfer buffer containing 5.8 grams of tris base, 2.9 grams of glycine, 0.37 grams of SDS, and 200 milliliters of methanol.
To transfer the size separated proteins from the polyacrylamide gel to a nitrocellulose membrane, assemble the sponges, filter papers, gel, and nitrocellulose membrane according to Western transfer protocol and run at 70 volts for one hour. After running the gel, incubate the nitrocellulose membrane in a protein solution of 1 to 5%bovine serum albumin or milk protein in 50 milliliters of TBST buffer for one hour to prevent non-specific protein binding. Wash the membrane three times using TBST buffer to remove any unbound blocking solution.
Next, incubate the membrane for one hour with the diluted horseradish peroxidase linked antibody specific for histidine tagged protein. Then wash the membrane three times in TBST buffer to remove any unbound antibodies. For digital imaging, make a solution of equal parts of chemiluminescent substrate solutions, 750 microliters each in a 1.5 milliliter tube, and incubate the membrane for at least five minutes in the solution.
Place the membrane protein side down on the blot scanner and run the acquisition software to quantify the protein in both the the pellet and supernatant fractions. To acquire the data, start the quantitative signal measurements using the acquisition software with the blot scanner. Normalize all quantitative measurements in the supernatant and pellet fractions to the total protein loaded.
In the saturating binding experiment, plot the percentage of protein bound versus amylopectin concentration and fit the data using data analysis software to calculate the dissociation constant, Kd.The binding capacity of SEX4 to ConA-sepharose amylopectin beads was analyzed using SDS page. The presence of SEX4 protein in the pellet fraction and bovine serum albumin in the supernatant fraction was noted. W278A, an known SEX4 mutant with significantly reduced glucan binding ability, appeared in the supernatant fraction, indicating the utility and specificity of this assay.
Western blotting analysis revealed a significant increase in the detection of SEX4 and the method is specific for detecting glucan phosphates with an end terminal histidine tag. The cosedimentation assay was tested at three different SEX4 concentrations. While two micrograms of SEX4 is sufficient to visualize through chemiluminescence detection, using five or 10 micrograms of SEX4 allows for more accurate detection.
Also, increased SEX4 binding with increased amylopectin concentration was noted. The binding affinity of amylopectin and SEX4 wild type protein at different concentrations was determined and the percent protein bound data to the specific one site binding model gave a Kd of approximately 1.03 milligrams per milliliter. The applicability of this method to measure the binding of laforin, LSF2, corn SEX4, and potato SEX4 against potato amylopectin was assessed.
The results demonstrated that the tested proteins from agronomically important crops are also differentially bound to amylopectin. It is crucial to remember the normalization of all quantitative measurements in the pellet and supernatant fractions to the total protein loaded. To ensure all amylopectin is bound to the beads, save the supernatant fractions to perform the deep glucose assay later.
Glucan phosphatase is employed distinct mechanisms to bind, locate, and dephosphorylate carbohydrates. This method allows for the exploration of glucan phosphatase's binding to different structural domains within large polymeric complexes for position specific dephosphorylation.
