Question
Please show your work! Thanks!
1. Use the imaginary gel mages below to answer the questions below. Use the molecular weight standards (in KDo) to estimate sizes (do not graph). three gels were looded with the same three purified proteins B.cand D (loaded in lones B,cond b) Gel 1 was run under non-reducing conditions (SDS) Gel 2 was run under reducing conditions (sDs and 6el 3 was run native (assume this tells the molecular weight in the native state) Use these images to answer the questions below. Ge 3 A Gel 2 50,7 355 288 la. Using all three gel images if the bond in Gel1.Lane B is at 120 KDa, estimate the number and size of the subunits making up the native protein. lb. Using all three gel images, if the bands in Gel 2, Lane Core at 20 and 45 KDa, estimate the number and size of the subunits making up the native protein. 1c, using all three gel images, if the band in 6el 2.Lane D is at 30 KDa, estimate the number and size of the subunits making up the native protein. 1d. What type of bonds or forces hold the subunits together in the protein in Ione B? (circle one) disulfide hydrogen bonds probably both neither, there is one subunit le. What type of bonds or forces hold the subunits together in the protein lone D? one) in (circle hydrogen bonds probably both disulfide neither, there is one subunit lf. Define the protein in lone B using the following system: homodimer, heterodimer,homotrimer. etc. Protein B is a:
Explanation / Answer
1a. Gel 1 is under non-reducing conditions, thus shows that protein in Lane B has a total mol. wt. of 120 kDa. However, it shows two bands when run under non-reducing conditions in gel 2. Thus, from the marker, we can conclude, the protein has two subunits of 50 + 70 kDa.
1b. From gel 1 and 2, we can conclude that protein in lane C has a subunit of 20 kDa and two more subunits of 45 kDa each, which total upto 90 kDa as seen in gel 2. This is confirmed by a 110 kDa band seen in gel 3, run under native conditions.
1c. Protein in lane D has mol wt of 60 kDa with two subunits of 30 kDa each. This is because under native gel 3, the band corresponds to 60 kDa.
1d. Disulfide bonds hold together the two subunits of protein in lane B. Disulfide bonds can be broken down by -mercaptoethanol, thus, in gel 2, lane B shows two bands.
1e. Disulfide bonds hold together the two subunits of protein in lane D. Disulfide bonds can be broken down by -mercaptoethanol, but the two subunits being of same size, run at the same place on gel 2.
1f. Protein B is a – heterodimer (because the two subunits are of different sizes).