There are various techniques through which proteins can be identified. The aim is to identify all proteins and make proteome map. To study particular disease we have required quantity of protein. Given below are the some techniques used to identify all proteins.
Every protein has different antibodies. Specific antibody will bind with the specific protein. Antibodies are bind with the beads and then proteins bind with the specific antibody.
• This technique is very sensitive.
• In living cells proteins interact, but antibodies must bind with desired protein without separation techniques.
• Antibodies of limited protein have been identified.
• For a protein to be identified by antibody its protein must be known.
• If we have to find a novel protein this technique cannot be used.
• It is very costly, chips are very expensive.
• Different antibodies are required to identify each protein for example, a panel of anti bodies could be used to identify all the different proteins in the proteome but this could required as many antibodies as these were target proteins including specific antibodies for different post-transcriptional variants of the same protein.
Through Affinity Chromatography
It is a selective (specific to study function and interaction of protein) technique in which binding affinity is exploited. We attach any component with the beads, those components which specifically bind with the desired protein. For example, concavalin A (specifically binds with glucose).
Three step process:
Step 1: Covalently attach X-molecule e.g. glucose to the beads or to the column.
Step 2: Adding a mixture of protein to the column then wash the column with buffer to remove unbound proteins except concavalin.
Step 3: Eluting the desired protein by adding a high concentration of a soluble form of X-molecule or altering the conditions to decrease binding affinity (changing of temperature and pH).
Through Antibody Array
Antibody has a structure called "Paratope" while antigen has a structure called "Epitope". Paratope works like a key and epitope works like a lock. Binding takes place between paratope and epitope. Identification of protein through antibody array involves the following procedure:
• Take a glass chip.
• Mount (covalently attach) on it different antibodies.
• At least 100 antibodies will attach.
• Specific epitope will bind with antibody.
Suppose pH given is 7 and temperature is 25°C, it is not necessary that all the antibodies bind with the proteins. Antibodies have different pH and temperature range. Those proteins which will fulfill all these conditions will only bind with its antibody.
About Author / Additional Info: