In your laboratory, you plan to use ion-exchange chromatography to separate the peptide shown below from...
In your laboratory, you plan to use ion-exchange chromatography to separate the peptide shown below from a mixture of different peptides at pH 7.0. Should you choose a matrix containing DEAE (+ charge) or CM (- charge) groups?
GLEKSLVRLGDVQPSLGKESRAKKFQRQ
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In your laboratory, you plan to use ion-exchange chromatography
to separate the peptide shown below from...
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography....
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography. IDH will be eluted from the column using increasing concentrations of KCI. IDH will be tracked using the IDH activity assay and the extent of purification will be analyzed using SDS PAGE Last week we assayed for the activity of the enzyme, isocitrate dehydrogenase, from E.coli strains carrying a plasmid that over-expresses the E. coli IDH gene. To purify proteins from cel extracts, biochemists...
I want to separate a protein from a crude yeast extract by ion exchange at pH...
I want to separate a protein from a crude yeast extract by ion exchange at pH 7.4. Should I use a DEAE column or CM cellulose column? The isoelectric point (pI) of the protein is greater than the pH (pI = 9). Explain.
How would you use ion exchange chromatography to seperate a mixture of alanine, glutamic acid, and...
How would you use ion exchange chromatography to seperate a mixture of alanine, glutamic acid, and lysine if you wished to maintain the pH at 7.5 and use only a single ion exchange column ( either an anionic or cationic exchanger). Describe the expected order of elution under the conditions which you propose.
Q1: You have two short peptides: Peptide A) KVDFSA; Peptide B) IRGTKL a. Write the chemical...
Q1: You have two short peptides: Peptide A) KVDFSA; Peptide B) IRGTKL a. Write the chemical structure of peptide A as it would appear at pH 7. Include all charges and begin from the N-terminus b. determine the net charge on each of the peptides at pH 2, 7, and 11 c. You would now like to separate the two peptides. You decide to use a cation exchange column at pH 7 A cation exchange column is composed of negatively...
5. You have synthesized two different peptides to test for their use as drug inhibitors. However,...
5. You have synthesized two different peptides to test for their use as drug inhibitors. However, you accidentally mixed the peptides together and need to figure out a way to separate them back apart. Using the provided table of pKa values, determine the charge (if any) on each amino acid at pH 7.0, then describe how you could chromatographically separate the two peptides at pH 7.0. (8 points) Peptide A = SMIENKRKDP Peptide B = AELRAQDKEQ
NOTES: To separate on a size-exclusion column, differences in MW need to be greater than 10%....
NOTES: To separate on a size-exclusion column, differences in MW need to be greater than 10%. To separate proteins by charge, the difference in pI must be at least 0.5 pH unit. Consider the table of four proteins below: Protein Size pI Size exclusion Charge at pH 7.4 DEAE-elution 1 49 kDa 6.8 2 51 kDa 8.1 3 53 kDa 6.4 4 99 kDa 8.3 5 106 kDa 3.8 6 143 kDa 7.9 (a) In what order would the proteins...
4. You plan to separate a mixture of the two compounds below using a column chromatography...
4. You plan to separate a mixture of the two compounds below using a column chromatography normal conditions, which of the compounds would you expect to be eluted off the column first? Expla
(3 points) A Student uses cation exchange chromatography to separate Protein 1 pi9.5) and Protein 2...
(3 points) A Student uses cation exchange chromatography to separate Protein 1 pi9.5) and Protein 2 pl 45). The column is equilibrated using a 20m phosphate buffer, pH 6.0 and then the sample containing Protein 1 and Protein 2 is applied to the column. After the sample is applied to the column, the column is first washed with a low salt buffer. Subsequently, the column is washed with a high salt buffer. The elution profile is shown below. Clearly indicate...
4. (Challenge) You are purifying a protein from the brain of patients with an unusual type of...
4. (Challenge) You are purifying a protein from the brain of patients with an unusual type of dementia triggered by a coronavirus and you hypothesize that this protein may be a signature molecule of this type of dementia. To pursue this hypothesis, you collect brain tissue from patients post mortem, do differential centrifugation, density gradient centrifugation, CM (carboxymethyl cellulose) ion exchange chromatography and gel filtration. You then assess the purity of your protein using both native and SDS gel electrophoresis. The...
You develop a scheme to purify the protein RANIN, and use your "sequencing toolbox" to identify...
You develop a scheme to purify the protein RANIN, and use your "sequencing toolbox" to identify it. You suspect RANIN has quaternary structure, but the polypeptides are not separated by extreme pH. Suggest another method SDS-PAGE samples run with and without reducing agent tefis you RANIN does not have intermolecular disulfide bonds, but it is made up of two distinct polypeptide chains. Draw the SDS-PAGE results and explain how you know. RANIN chain A has a pi of approximately 9,...
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography. IDH will be eluted from the column using increasing concentrations of KCI. IDH will be tracked using the IDH activity assay and the extent of purification will be analyzed using SDS PAGE Last week we assayed for the activity of the enzyme, isocitrate dehydrogenase, from E.coli strains carrying a plasmid that over-expresses the E. coli IDH gene. To purify proteins from cel extracts, biochemists...
Q1: You have two short peptides: Peptide A) KVDFSA; Peptide B) IRGTKL a. Write the chemical structure of peptide A as it would appear at pH 7. Include all charges and begin from the N-terminus b. determine the net charge on each of the peptides at pH 2, 7, and 11 c. You would now like to separate the two peptides. You decide to use a cation exchange column at pH 7 A cation exchange column is composed of negatively...
5. You have synthesized two different peptides to test for their use as drug inhibitors. However, you accidentally mixed the peptides together and need to figure out a way to separate them back apart. Using the provided table of pKa values, determine the charge (if any) on each amino acid at pH 7.0, then describe how you could chromatographically separate the two peptides at pH 7.0. (8 points) Peptide A = SMIENKRKDP Peptide B = AELRAQDKEQ
4. You plan to separate a mixture of the two compounds below using a column chromatography normal conditions, which of the compounds would you expect to be eluted off the column first? Expla
(3 points) A Student uses cation exchange chromatography to separate Protein 1 pi9.5) and Protein 2 pl 45). The column is equilibrated using a 20m phosphate buffer, pH 6.0 and then the sample containing Protein 1 and Protein 2 is applied to the column. After the sample is applied to the column, the column is first washed with a low salt buffer. Subsequently, the column is washed with a high salt buffer. The elution profile is shown below. Clearly indicate...
You develop a scheme to purify the protein RANIN, and use your "sequencing toolbox" to identify it. You suspect RANIN has quaternary structure, but the polypeptides are not separated by extreme pH. Suggest another method SDS-PAGE samples run with and without reducing agent tefis you RANIN does not have intermolecular disulfide bonds, but it is made up of two distinct polypeptide chains. Draw the SDS-PAGE results and explain how you know. RANIN chain A has a pi of approximately 9,...
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