Log in
Biology
1
answer
0
watching
76
views

6. It is your desire to purify the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the common sparrow. The enzyme has a MW of ~35kDa and a theoretical pI of ~9.00. In addition the enzyme carries out the following reaction: Glyceraldehyde-3-phosphate + NAD+ + Pi ↔1,3 bisphosphoglycerate + NADH

a. Based on this information would a Q-sepharose column be a good ion-exchanger for this protein (1 point)? Why or why not (1 point)?

b. Would you expect this protein to bind to a HiTrapTM Blue HP column? Why or why not (1 points)?

c. If you wanted to do an enzymatic activity assay on this protein using the absorbance properties of NAD+/NADH at 340 nm, what substrate would you put in the reaction mixture: Glyceraldehyde-3-phosphate or 1,3-bisphosphoglycerate (2 points)? Why (2 points)? (NOTE: You want absorbance at 340 nm to increase throughout the assay)

d. The last step of your purification protocol involves running your protein sample over a size exclusion column. At first you choose a column with a molecular weight cutoff of 80 kDa. Due to the size of your protein you expect it to come off somewhere in the middle of the run and foolishly decide to start collecting fractions half-way through the run. When you come back you see your data and realize the protein came off in the “void” volume (very early in the run) suggesting that your protein is much larger than 80 kDa. What is a possible explanation for these data? (1 point)

Watch
1
answer
0
watching
76
views

For unlimited access to Homework Help, a Homework+ subscription is required.

Lelia Lubowitz
Lelia LubowitzLv2
28 Sep 2019

Unlock all answers

Get 1 free homework help answer.
Already have an account? Log in
Share

Related textbook solutions

Molecular Cell Biology
8 Edition,
Lodish
ISBN: 9781464183393
Biology: Science for Life with Physiology
6 Edition,
Belk
ISBN: 9780134555430
Concepts of Biology
OER Edition,
Openstax
ISBN: 9781938168116
Biology
2nd Edition, 2013
Openstax
ISBN: 9781947172517
Essentials of Biology
5 Edition,
Mader
ISBN: 9781259660269

Related questions

In class we expressed and purified the human form of LDH. Our purification protocol began with the Ni-affinity column. However, as you should have all noted, some significant containments still remained in our “pooled” sample. To remove these impurities we could have gone on and ran other chromatography columns such as ion exchange, size-exclusion columns, and other affinity columns. Given this, along with the sequence of LDH below, answer the following questions: 1 MATLKDQLIY NLLKEEQTPQ NKITVVGVGA VGMACAISIL MKDLADELAL VDVIEDKLKG 61 EMMDLQHGSL FLRTPKIVSG KDYNVTANSK LVIITAGARQ QEGESRLNLV QRNVNIFKFI 121 IPNVVKYSPN CKLLIVSNPV DILTYVAWKI SGFPKNRVIG SGCNLDSARF RYLMGERLGV 181 HPLSCHGWVL GEHGDSSVPV WSGMNVAGVS LKTLHPDLGT DKDKEQWKEV HKQVVESAYE 241 VIKLKGYTSW AIGLSVADLA ESIMKNLRRV HPVSTMIKGL YGIKDDVFLS VPCILGQNGI 301 SDLVKVTLTS EEEARLKKSA DTLWGIQKEL QF*

a. What is the theoretical pI of human LDH? Given this pI would a Qsepharose column be a good choice for further purification? Why or why not?

b. Would you expect LDH to bind to a HiTrapTM Blue HP column? Why or why not?

c. A typical last step of a purification protocol involves running your protein sample over a size exclusion column. Let’s say we do this for LDH. At first you choose a column with a molecular weight cutoff of 80 kDa. Due to the size of your protein you expect it to come off somewhere in the middle of the run and foolishly decide to start collecting fractions halfway through the run. When you come back you see your data and realize the protein came off in the “void” volume (very early in the run) suggesting that your protein is much larger than 80 kDa. What is a possible explanation for this data?

Biochemistry Protocols: Your First Protein Purification

As the newest student in a biochemistry research lab, you are given responsibility for purifying a mitochondria protein. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answers.

(a) You pick up 20 kg of beef hearts from a nearby slaughterhouse. You transport the hearts on ice, and perform each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart tissue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 7.2.

Why do you use beef heart tissue, and in such large quantity?

What is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? What happens to the tissue when it is homogenized?

(b) You subject the resulting heart homogenate, which is dense and opaque, to a series of differential centrifugation steps. What does this accomplish?

(c) You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, You centrifuge the solution, decant the supernatant, and discard the

pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet because it contains

the protein of interest. What is the rationale for the two-step addition of the salt?

(d) You solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze it overnight against large volumes of buffered (pH 7.2) solution. Why isn’t ammonium sulfate included in the dialysis buffer? Why do

you use the buffer solution instead of water?

(e) You run the dialyzed solution over a size-exclusion chromatographic column. Following the protocol, you collect the first protein fraction that exits the column, and discard the rest of the fractions that elute from the column later. You detect the protein by measuring UV absorbance (at 280 nm) in the fractions. What does the instruction to collect the first fraction tell you about the protein? Why is UV absorbance at 280 nm a good way to monitor for the presence

of protein in the eluted fractions?

(f) You place the fraction collected in (e) on a cation exchange chromatographic column. After discarding the initial solution that exits the column, you add a washing solution of higher pH to the column and collect the protein fraction that immediately elutes. Explain what you are doing.

(g) You run a small sample of your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharp bands. According to the protocol, the protein of interest is the one with the pI of 5.6, but you decide to do one more assay of the protein’s purity. You cut out the pI 5.6 band

and subject it to SDS polyacrylamide gel electrophoresis. The protein resolves as a single band. Why were you unconvinced of the purity of the “single” protein band on your

isoelectric focusing gel? What did the results of the SDS gel tell you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing.

1. Joshua used gel filtration chromatography to purify protein Z from crude homogenate prepared from Arabidopsis plants. He noticed that protein Z appeared to have eluted from the column in two separate peaks. Using a standard curve, he calculated the MW of protein Z eluted in the first peak to be 156 KDa, and the protein Z eluted in the second peak had the apparent MW of 78 KDa. Which one do you think is the correct MW? What does this tell you about the structure of the protein?

2. Read the following paragraph and correct all errors (4.15 points).

An unemployed high school student, Summer, has isolated a protein from a juniper tree with an overall negative charge. He performs ion exchange chromatography using a cationic resin with blue dextran to aid the proteins association with the column. As the proteins come off the column, the spec shows 4 peaks, only the first peak (wash peak) shows activity in an enzyme assay. Since all fractions associated with this peak had activity, they were pooled and total protein assayed. Total protein analysis was assayed using Bicine reagent which interacts with proteins to cause a change in absorption when measured with a spectrophotometer. The results of the total protein calculation showed that there was a modest decrease in total protein which means that there was a large amount of pure protein of his interest present in the sample.

3.You have accidentally mixed a bunch of amino acids (tryptophan, tyrosine, alanine, selenocysteine, proline, arginine) together and once your boss finds out, you will be fired on the spot. Your only hope of saving your job is to separate them before anyone finds out. You decide that the only viable option is to use ion exchange chromatography. Explain, in detail, how you would do this? (4.15 points)

4. Why should you not lose any LDH in your 20k x g spin? How does this explain an increase the fold purification of LDH in your 20k x g sample compared to your crude homogenate? (2 points)

Weekly leaderboard

Home
Homework Help 3,900,000
Biology 460,000
Start filling in the gaps now
Log in