题目
单项选择题
Which technique comes right before a western blot, according to the video?
选项
A.agarose gel electrophoresis
B.determining protein concentrations using a standard curve
C.SDS-PAGE
D.separating proteins by relative hydrophobicity
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标准答案
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思路分析
Question: Which technique comes right before a western blot, according to the video?
Option 1: agarose gel electrophoresis. This method is typically used for separating nucleic acids (DNA/RNA) or very large protein complexes in some contexts, but when preparing proteins for a Western blot, the common gel used is polyacrylamide (SDS-PAGE), not agaro......Login to view full explanation登录即可查看完整答案
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类似问题
Shapiro et al. (1967) noted that SDS-PAGE separation could be used for a wide variety of proteins, including those with multiple subunits, those with disulfide bonds, and those with a wide range of pI values, where low pI indicates high proportion of acidic residues and high pI indicates high proportion of highly basic residues. They said that "despite the choice of a group of proteins with a range of isoelectric points from 4 to 11... all points approximated the fitted curves, suggesting that SDS minimizes the native charge differences and that all proteins migrate as anions as the result of complex formation with SDS. The extensive disruption of hydrogen, hydrophobic, and disulfide linkages by SDS and [beta-mercaptoethanol] results in the quantitative solubilization of many insoluble proteins. These factors and the ease of the polyacrylamide technique strongly recommend it as the electrophoretic method of choice." It must have seemed quite surprising that SDS would be so effective at overwhelming the inherent and variable charge that proteins have! Review SDS structure here Links to an external site. if needed. All of the following might be interactions that SDS makes with proteins as it helps to unfold and coat them except:
SDS-PAGE is an extremely common protein separation technique that you might have come across before. You might have performed it or might have heard it mentioned. You might not realize how old it is - the separation method was published in 1966 and then a follow-up article in 1967 (Shapiro et al.) described how it can be used to estimate/confirm protein size. In the image below Shapiro et al. (1967) showed in their article how the technique could be used to estimate protein size, by showing how it worked on proteins of known size. Note that the y-axis refers to 'molecular weight divided by 1000', also commonly known as 'kilodaltons' in protein work: In the inset graph (top right) they show untransformed data. Relative migration is on the x-axis, and the protein 'Lys' for 'Lysozyme' migrated down to the bottom of the gel so it's at 100%. It is known to have a very small molecular weight of about 15 kDa so it is very low on the y-axis. Meanwhile the albumin known as BSA ('Bovine Serum Albumin') is much larger: a monomer is 66 kDa so a dimer is 132 kDa. It only migrated about halfway down the gel. The larger graph on the left shows that the relationship between migration and kDa is not linear unless the y-axis is log-transformed (using log base 10). The relationship is not as clear at low molecular weights. Which one of the following best indicates a likely reason for performing the log(10) transformation? Why are there two graphs? Why wasn't the one on the right sufficient?
What causes the proteins to have a similar mass:charge ratio to each other?
To identify the green fluorescent protein on the SDS-PAGE gel, we were trying to
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