Passage
The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting. In addition, the protein insulin receptor substrate 4 (IRS4) , known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4. Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection. HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively) . Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away. The bound proteins were then visualized by Western blot using α-myc antibodies. The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb) . Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1.
Figure 1 Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub) . The results are shown in Figure 2.
Figure 2 Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control) , myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid
Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95.
-Which of the following characteristics do the flag and myc tags have in common? They have the same:

Question

Passage
The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting. In addition, the protein insulin receptor substrate 4 (IRS4) , known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4. Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection. HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively) . Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away. The bound proteins were then visualized by Western blot using α-myc antibodies. The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb) . Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1.

Figure 1 Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub) . The results are shown in Figure 2.

Figure 2 Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control) , myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid
Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95.
-Which of the following characteristics do the flag and myc tags have in common? They have the same:

Quizplus · Accepted Answer

11ecba2d_0efd_2b0e_a3bf_e564a0e7ba2c_MD0008_00 The properties of peptides and proteins are determined by their amino acid composition. Each of the 20 canonical amino acids contains a unique side chain that may be polar or nonpolar. Polar amino acids can be further classified as acidic (negatively charged at physiological pH), uncharged, or basic (positively charged). The net charge of a peptide or protein is the sum of the charges of its side chains and the charges of the N- and C-termini. At physiological pH, the N-terminus is positively charged and the C-terminus is negatively charged, and the two charges cancel, leaving only the side chain charges. The passage gives the amino acid sequences of both myc (EQKLISEEDL) and flag (DYKDDDDK). At physiological pH, the myc peptide contains four negatively charged residues: three glutamate residues (E) and one aspartate residue (D). It also contains one positively charged residue (lysine, K). Accordingly, the net charge is −3 (−4 + 1 = −3). The flag peptide has five negatively charged aspartate residues and two positively charged lysine residues, also yielding a net charge of −3 (−5 + 2 = −3). Therefore, flag and myc have the same net charge. (Choice A) The myc peptide has three nonpolar (hydrophobic) residues: two leucines (L) and one isoleucine (I). The flag peptide contains no nonpolar residues. (Choice B) Antibodies are highly specific and typically bind a single chemical group, called an epitope. Therefore, an antibody that binds flag (α-flag) would most likely not bind myc. (Choice C) The flag peptide has five acidic residues (aspartate, D), whereas myc has only four (three glutamate, E, and one aspartate, D). Educational objective: Different peptides and proteins have different properties based on their amino acid composition. At any given pH, the net charge of a peptide is the sum of the charges of its side chains and the N- and C-termini. At physiological pH, the positive charge of the N-terminus cancels the negative charge of the C-terminus.

Passage The Ankyrin Repeat and SOCS Box Containing Protein 4 (Asb-4)