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Suppose you are purifying a protein on a weak cation exchanger(pH 7.5) and it comes off at 300mM salt. predict what saltconcentraiton (lower, same, higher) the protein would come off ifyou used a strong cation exchanger. Explain the logic behind yourprediction in terms of the differences between a weakand strong exchanger.

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Related questions

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.

Please I need help answering those questions i always lose bunch of points i'm not doing it correctly.

Use information from Nature Reviews Microbiology to answer the following question.What is the role of EF-G and EF4 in protein synthesis? Under what circumstances does EF4 participate in translation and what is its role?

from this reading The Highly Conserved LepA Is a Ribosomal Elongation Factor that Back-Translocates the Ribosome Qin Y,Polacek N, Vesper O, Staub E, Einfeldt E, Wilson D.N, and Nierhaus N.H. Cell 127: 721-733 (2006).

Q2. In which species is Lep A found and in which ones is it absent? Is the result expected based on your knowledge of the endosimbiosis theory?

Q3.

3a What is the percentage of identity between LepA and its ortholog genes?

3b. (5pts) Indicate what domains are conserved between Ecoli EFG and LepA, and which ones are not.

3c. What do grey and black boxes in the sequence alignment indicate?

Q4. Analysis of in vivo experiments shown in Figure 3. Only analyze graph on the left side of the figure.

4a. What is the difference between the bacteria whose growth are shown by the blue and the green curves? What can be concluded about the effect of EFG on bacterial growth?

4b. Which protein is expressed by bacteria whose growth is shown by the yellow curve? Does the expression of this protein affect bacterial growth? Compare the yellow and blue curves. How can this result be explained?

4c. Explain why the addition of IPTG results in lower bacterial growth. Compare the yellow and red curves?

4d. Why is the green curve an important control of the experiment? The green curve.

The next questions are based on the reading of the article Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations.

Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations Pech M, Karim Z, Yamamoto H, Kitakawa H, Qin Y, and Nierhaus K. PNAS 108: 3199-3203(2011)

Studies done in E. coli using a knockout mutant of the lepA gene (ΔlepA , lepA gene has been deleted) did not show any phenotype when grown in rich LB medium. This result was unexpected due to the high level of conservation of EF-4 among species.

Q5. Equal amounts of wild type and mutant cells (lacking the lepA gene) were mixed and co-cultured for 24h. The population distribution was determined every 4 hours. Figure 1 indicates changes in the % of mutant cells over several generations.

Q5a. What is the question that researchers addressed in the experiments shown in Figure 1?

Q5b. Which curve represents the control of the experiment? Do the two strains grow at similar rate? How do you know?

Q5c. Do high salt concentration and/or low temperature have any effect on the growth of mutant cells?

Q5d. What can you conclude about the role of LepA on bacteria growth? When is this protein important?

Q6.

6a. (6pts) Where in the cell is EF4 located? Did you expect that this would be the EF4 location? Why or why not?

What is the question that the researchers want to address in the experiment shown in Figure 2?

6c. Analyze the green and red bars graph shown in Figure 2. What technique was used to detect EF4? Is there any change in EF4 localization?

Q7. Summarize the main points of the model shown in Figure 4

For this assignment, we will consider a protein expressed in theliver of an African cheetah (Acinonyx jubatus). Biochemistsdetermined that this protein is involved in the production ofglycogen.

In a stunning announcement, a snowboarder in Antarctica claimsto have found a new species of cheetah that is able to survivefreezing temperatures and a long, dark winter. The snowboarderdescribed a slow, fat, white-furred cheetah that may be related tothe African cheetah. Scientists isolated a fragment of DNA from theobese feline creature which codes for the liver protein:

(3' end) ...CTACGTTCAGCTTGTCACGACTGGTTA... (5' end)

(5' end) ...GATGCAAGTCGAACAGTGCTGACCAAT... (3' end)

At almost the same time, a sleek, fast, white-coated cheetah wasfound in the Andes in South America where cheetahs were not knownto exist. With some difficulty, a geneticist convinced the cheetahto allow her to swab the inside of the big cat's cheek to get asample of DNA. Soon after, the geneticist was able to isolate thefollowing DNA fragment believed to be involved with glycogenproduction in the liver:

(3' end) ...TCACCTACGTTCAATAGGTCACGACTGG... (5' end)

(5' end) ...AGTGGATGCAAGTTATCCAGTGCTGACC... (3' end)

The original African cheetah DNA fragment describing the liverprotein in question is this:

(3' end) ...CACCTACGTTCAGGAGGTCAGGACTGGTTA... (5' end)

(5' end) ...GTGGATGCAAGTCCTCCAGTCCTGACCAAT... (3' end)

You are part of a team of geneticists, evolutionary biologists,and bioinformatics experts. You will attempt to describe thedifferences between the three species of cheetah in both geneticand evolutionary terms.

1)For each DNA sequence, transcribe to mRNA and then find thechain of amino acids that it produces. Remember that a gene mustbegin with the "Start" codon.

2)Describe the differences in the basic genetic code between thethree species. How many DNA point mutations would be required tochange from one species to the others? How do the amino acidsequences of the proteins differ? Are there any silent mutationsthat may not effect the function of the protein?

3)Looking at the amino acid substitutions that exist between thespecies, would you expect there to be large functional differencesbetween the two slightly different proteins based on the propertiesof the substituted amino acids? Explain why you think this might bethe case.

4)Quantitatively assess the evolutionary distance between eachpair of the three species using the substitution matrix and PAMscores. (This involves three pairs: African/Andes,African/Antarctic, and Antarctic/Andes.)

5)Propose a scenario describing how the two new cheetah speciesmight have evolved from the African cheetah. Were the two newcheetahs once a single species which then branched to form twoseparate species? Or did they evolve independently from the Africancheetah? Be sure to include in your scenario how the new cheetahsmanaged to move from Africa to their present locations. Estimate arelative time line for each separate speciation event. (Aspeciation event is the genetic or environmental event that resultsin the creation of a new species.) How might the three proteinsexplain the survivability of the three species in their nativehabitat? (A little speculation is OK here. You are presenting ahypothesis based on the available evidence. Further research wouldbe required to prove your hypothesis.)

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