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Describe the ten steps involved in making a recombinant protein using methods in biotechnology.
1) Determine the protein of interest - Here is where you do research on what protein is needed for a specific disease state.
2) Characterization of the protein - Here is where you find the amino acid sequence of the protein.
3) Finding the protein’s gene - Here is where you search for the protein’s gene sequence. This is usually done via a library plasmind.
4) Cloning the protein’s gene
5) Expressing the gene in an organism - This is done via transfection or transformation.
6) Optimizing the expression in culture - Production is acheived.
7) Harvesting the protein from the cell culture - The protein must be active, so in order to be considered successful, the protein must undergo all of the necessary post-translational modifications needed to activate it.
8) Purifying the protein - The protein must not contain any residues that may induce an immunological response.
9) Packaging and dispensing - The protein’s stability will determine whether or not it is refrigerated.
10) Administering the recombinant protein - Here is where the protein is administered as a drug, i.e. insulin injections.
Name nine lab techniques that can be used to characterize proteins.
- 1) Gel Electrophoresis
- 2) Native Gel Electrophoresis
- 3) Southern Blot
- 4) Northern Blot
- 5) Western Blot
- 6) Restriction Fragment Length Polymorphisms (RFLP)
- 7) Mass Spectroscopy
- 8) DNA and RNA Electrophoresis
- 9) DNA Probing via hybridization
What are three things that lab techniques can identify in protein characterization?
- 1) size
- 2) composition of amino acid
- 3) sequence of amino acid
Describe Gel Electrophoresis.
The protein in question is ran through a gel. Its size determines how far it travels, the largest running the shortest distance. This technique involves the denaturing of the protein via (sodium dodecyl sulfate-polyacoylamine gel electrophoresis) SDS-PAGE. This is done so that the protein is no longer folded and the amino acid sequence may be determined in another method. This technique is ran to determine either the size or number of protein.Note: The size of the dot determines the quantity of the protein, while the distance it travels determines the molecular weight.
Describe Native Gel Electrophoresis.
Like Gel Electrophoresis, this lab technique runs the protein through a gel without the use of SDS-PAGE to denature the protein. This being the case, molecular weight cannot be determined by this technique, but the size and shape of the active protein can be. A protein monomer may consist of one band that runs further while a protein dimer consists of a dimer which runs a shorter distance.
Describe Mass Spectroscopy.
This is a lab technique in which the amino acid sequence/fingerprint is found. The gel is ran through MALDI-MS which processes the amino acid composition of the protein.
Describe Western Blot.
This lab technique is similar to the Gel Electrophoresis in which the protein is ran through a gel via SDS-PAGE, which cuases it to denature. It is then transferred to a membrane and then probed with an antibody which is tagged with either a fluorescent protein or other means of identifying the antibody. This is done to determine a protein’s molecular weight or its identification.
Describe DNA and RNA Electrophoresis.
This lab technique doesn’t study protein, but DNA/RNA. Instead of using SDS-PAGE to denature, the DNA strand (not RNA, because it is already single-stranded) is melted. The fragments are then ran on a gel, where the smaller fragments will run further. This is done to determine the DNA fragment size, and its number.
Describe DNA probing.
This lab technique is done by taking a fragment of DNA sequencing and matching up its bases with that of a single DNA strand or an mRNA strand. Its target is the coding strand. It has the same sequence as the template strand. Note: The DNA probing is considered a Southern Blot while the mRNA probing is referred to as a Northern Blot.