experiment 6: SDS-PAGE of rGFP fractions Flashcards
what is the main purpose behind a SDS-PAGE gel?
it separates proteins based on their sizes (molecular weights) to estimate relative molecular weight by comparing it to a MW standard (MW ladder) and can be used to estimate the relative amount of a particular protein in a mixture of proteins (purity)
what is the underlying principle behind SDS-PAGE gel?
separation of proteins is based on size. SDS neutralizes the charge on the proteins and gives all proteins present an equal charge-to-mass ratio. SDS, along with heat, denatures the proteins. a reducing agent (β-mercaptoethanol) is used to break disulfide bonds. a voltage is applied so that negatively charged proteins move towards the positive electrode. larger proteins move more slowly than smaller proteins.
what is the purpose of SDS in the system?
SDS has a polar negatively charged head group and a hydrophobic tail and functions as an ionic detergent that denatures the protein. SDS coats the proteins so that there is 1 molecule of SDS per 2 amino acid residues, giving proteins an equal charge-to-mass ratio. separation is therefore based only on size rather than net charge making the use of the MW ladder more accurate.
what is the purpose of the beta-mercaptoethanol in the sample-loading buffer?
it acts as a reducing agent to break the disulfide bridge holding the protein complex together
what is the purpose of the glycerol in the sample-loading buffer?
glycerol increases the density of the samples so that they will sink into the wells when they are loaded. glycerol also increases viscosity for easier sample loading as well as allowing the proteins to run down the gel at the same time.
what is the purpose of the bis-acrylamide?
it acts as a cross-linker that attaches the strings of acrylamide polymer to create a solidified gel. the higher the ratio of acrylamide to bis-acrylamide there is, the faster the reaction goes.
what is the purpose of the stacking gel?
the stacking gel has properties that cause the proteins in the sample to be concentrated into a narrow zone at the top of the resolving gel, permitting effective and reproducible separation of the SDS-coated proteins by size.
what is the main difference between the stacking and resolving gels?
the stacking gel is polymerized with a small percentage of acrylamide and bisacrylamide to ensure high porosity, and is buffered with Tris-HCI buffer at pH 6.8, whereas the resolving gel contains a higher percentage of acrylamide and is cast in Tris-HCI at a higher pH (8.8).
predict what would happen if SDS is left out of the system
separation by multiple independent variables would occur leading to inconclusive results and no protein denaturing (proteins would not have the same charge-to-mass ratio, since proteins are not denatured their shape could influence the distance travelled)
predict what would happen if β-mercaptoethanol is left out of the system
the intra-disulfide bridges would remain intact and the protein will not be completely linearized. this can affect the migration of the protein and the multiprotein complexes will be viewed as one protein complex with a high molecular weight, all leading to in an incorrect estimation of the MW.
predict what would happen if glycerol is left out of the system
less dense proteins will be higher in the well and denser proteins will be closer to the bottom of the well. therefore, not all of the proteins will begin running through the gel at the same time which may lead to inconclusive results, as the distance between the bands may not necessarily correlate to their respective molecular weights. the samples will also take longer to sink into the wells meaning it would take more time for bands to be visible.
predict what would happen if bis-acrylamide is left out of the system
the acrylamide will only form linear bonds with one another, and the gel will lack pores. because the pores are responsible for the sieving action, there will be nothing in the way to inhibit the movement of the proteins, and they may run off the gel. therefore, the results will be inconclusive
