Question

24, (8 pts) How would purify the RTML protein from all other components in a tissue: ...if you have no knowledge of this protein except that you know its molecular weight and suspect that it is overexpressed in a certain type of pancreatic tumor. a. Method- Centrifuge and then purity-how.? b. Theory - Seperation of protein from other tissue components to Move Casily allow puils ration c. Controls - Hay RTML proten to show as the normal but other components as absormed ....if you have introduced a modified RTML gene that expresses this protein with an "epitope tag" a. Method- Contribuer at the party b. Theory - Experten of epilcope will show distinction during separation c. Controls - Expressed RTML genc shown as normal other tissues should as abnormal.

Answer 1

A.If only known about molecular weight of protein then best method for purify the RTML protein by size exclusion chromatography.

B.Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is known as size exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel.

In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.

C. Using fluorescent tag or label the RTML protein can be detected by others components of cell.

• Enzyme-linked immunosorbent assay (ELISA): Specifically can detect protein down to pg/mL.
• Protein immunoprecipitation: technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
• Immunoelectrophoresis: separation and characterization of proteins based on electrophoresis and reaction with antibodies.

A.Purification of a tagged protein :- HPLC immunoprecipitation.

B.The way to tag proteins is to engineer an antigen peptide tag onto the protein, and then purify the protein on a column or by incubating with a loose resin that is coated with an immobilized antibody. This particular procedure is known as immunoprecipitation. Immunoprecipitation is quite capable of generating an extremely specific interaction which usually results in binding only the desired protein. The purified tagged proteins can then easily be separated from the other proteins in solution and later eluted back into clean solution.

When the tags are not needed anymore, they can be cleaved off by a protease. This often involves engineering a protease cleavage site between the tag and the protein.

HPLC:-

High performance liquid chromatography or high pressure liquid chromatography is a form of chromatography applying high pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved. The most common form is "reversed phase" HPLC, where the column material is hydrophobic. The proteins are eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity. After purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized. HPLC purification frequently results in denaturation of the purified proteins and is thus not applicable to proteins that do not spontaneously refold.

C.Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.