The overall goal of this procedure is to map the interaction between two proteins or protein domains. In this case, the cancer related protein domains still and CHFR are studied. The overall goal of this procedure is to map the interaction between two proteins or protein domains.
In this case, the cancer related protein domains still and CHFR are studied. The second step is to incubate the CHFR peptide array with its binding partner, the still protein. Next, the array is washed and incubated with horse radish peroxidase, conjugated antibody.
The array is then subjected to chem luminescence detection to detect the binding. The final step is to analyze the results and identify the binding sites between the proteins. This study paves the way for designing rational inhibitors of still CHFR binding, whose uncontrolled interaction can lead to the development of cancer.
This method can help answer key questions in the research of protein interaction, such as what are the specific regions that meditate An interaction between two proteins, Divide the sequence of the target protein into 10 to 20 residue peptides. That partially overlap. The amount of overlap can be varied, but in principle, the longer the overlap, the better.
When designing the peptides take into account known secondary structure elements in the protein that can be responsible for the interaction. The designed peptide array can be ordered through commercial vendors. For this experiment, peptides from the CHFR domain residues 4 24 through 6 62 are covalently bound to the array through the C terminus.
After receiving the designed array, prepare it for the experiment by first blocking it to prevent nonspecific binding. Make a blocking solution of 2.5%weight per volume. Skim milk powder in tris buffer solution containing 0.05%Tween 20 or TBST to prevent nonspecific binding.
Immerse the array in five milliliters of blocking solution. Incubate the array for two to four hours at room temperature or overnight at four degrees Celsius on a shaker. Wash the array first with five milliliters of blocking solution for 30 seconds.
Then wash the array twice with five milliliters of TBST for five minutes on a shaker at room temperature. Next, incubate the washed array with five milliliters of poly histidine tagged protein solution containing 2.5%weight per volume. Skim milk powder to prevent non-specific binding.
Here, a 4.5 micromolar solution of still protein residues 500 through six 50 is used. Then incubate the array in the protein solution for three to eight hours at room temperature or overnight at four degrees Celsius. Following incubation in the protein solution.
Wash the array with five milliliters of TBST for 30 seconds on a shaker at room temperature, followed by two additional five minute washes. Now incubate the washed array with five milliliters of diluted horse radish peroxidase conjugated antibody in an incubation buffer that contains the same ingredients at the same concentrations as the blocking solution for one hour on a shaker at room temperature, the antibody can bind either the target protein or the tag, perform control experiments, testing the interaction of the antibody with peptides on the array by repeating the same protocol on a separate array slide without incubating with the protein before reading the arrays. Wash them as before with five milliliters of TBST.
Carry out chemiluminescence development with an enhanced chemiluminescence western blotting substrate kit. Perform the detection with a luminescent image analyzer such as a Fuji film LAS 3000 camera. Analyze the results, making sure that both duplicates on the array show the same signals so the results are reliable.
If the structure of the protein partner is known, search on the structure for the binding peptides and look for the binding sites. Keep in mind that peptides that are far in the sequence can be together in the tertiary structure and create a binding site. In our example, still is a highly important Centro Somal protein that controls normal cell division and cell proliferation still interacts with several proteins through its intrinsically disordered region.
Using the designed peptide array, the still binding sites in the tumor suppressor CHFR were revealed. Each black spot in the array represents A-C-H-F-R derived peptide that bound to still note that each peptide appears twice since it exists in duplicate, which verifies the reliability of the results. The peptide array screening revealed seven CHFR derived peptides that bound to the still intrinsically disordered region residues 500 through six 50.
These results allowed us to map the binding site of still on CHFR. Despite the distance of these seven peptides on the CHFR primary sequence. They create a well-defined binding site for still in the CHFR tertiary structure.
This method is applicable for characterizing every protein protein interaction Following this procedure. Quantitative methods like fluorescence and isotope or ITC should be used in order to verify, in quantify the peptide introduction.
