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Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells. During FRAP, the fluorescence of a green fluorescent protein (GFP) -tagged molecule is first measured and then photodestroyed in targeted cell regions. Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region. The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins. HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure.
Figure 1 Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP. FRAP was performed on euchromatin and heterochromatin. Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively. The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1 Effect of HMGB1 and HMGA1 on H1-GFP Mobility
Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10) :4321-8.
-The molecules in Figure 1 were analyzed using gel electrophoresis, and the results are shown below. Which of the following describes the correct experimental methodology?

Question

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Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells. During FRAP, the fluorescence of a green fluorescent protein (GFP) -tagged molecule is first measured and then photodestroyed in targeted cell regions. Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region. The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins. HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure.

Figure 1 Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP. FRAP was performed on euchromatin and heterochromatin. Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively. The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1 Effect of HMGB1 and HMGA1 on H1-GFP Mobility

Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10) :4321-8.
-The molecules in Figure 1 were analyzed using gel electrophoresis, and the results are shown below. Which of the following describes the correct experimental methodology?

Quizplus · Accepted Answer

11ecba2d_0f3c_304b_a3bf_39cebef02b22_MD0008_00 The proteins shown in Figure 1 were analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which is used to separate proteins by size (molecular weight). During this procedure, samples are first heated with SDS, a denaturing detergent that coats proteins with a negative charge proportional to their length (size). SDS binds hydrophobic regions in the protein core, unfolding and extending the polypeptide into a linear arrangement of amino acids (ie, primary structure). This detergent also solubilizes the proteins and releases them from all associations with other molecules. Proteins are generally smaller than DNA molecules and are therefore loaded on a highly crosslinked polyacrylamide gel instead of an agarose gel (has larger pores). An electric current is applied and negatively coated proteins travel through the gel matrix and toward the anode, denoted as the positive electrode in electrophoresis. (Note that this convention is opposite that used in electrochemical cells, in which the anode is negative.) Smaller proteins travel faster than larger ones, creating lanes of size-separated protein bands. These bands are then stained for visualization using Coomassie blue or the more sensitive silver stain, both of which bind tightly to proteins. Band intensity is an indicator of protein abundance. (Choices A and B) The intrinsic protein charge is masked by SDS. When necessary, reducing agents (eg, β-mercaptoethanol) are used in SDS-PAGE to break disulfide bonds, disrupting tertiary and quaternary protein structure. (Choice C) Figure 1 shows the chromatin binding motifs of HMG proteins, not DNA. In agarose gel electrophoresis, ethidium bromide is used as a stain to make DNA bands (not proteins) fluoresce; this effect occurs as a result of ethidium bromide's ability to intercalate ("slip in") between DNA base pairs. Educational objective: SDS-PAGE is used to separate proteins by molecular weight. During the procedure, SDS coats proteins with a negative charge. An electric current is then applied, and smaller proteins travel through a polyacrylamide gel and toward the positive anode faster than larger ones, creating lanes of size-separated protein bands.

Passage Quantitative Fluorescence Recovery After Photobleaching (FRAP) Is Used to Study