* 1. Your Gel filtration column back pressure is very high, what should you do?

* 2. You loaded a protein sample to a Q column. The target protein (pI=6.0) somehow can not be eluted from the column using pH8.5 buffer containing 1 M NaCl, what should you do to elute this protein from column (very important: do not use harsh condition to destroy enzymatic activity). This protein will aggregate and lose enzyme activity at 1.1 M NaCl salt concentration and pH 8.5

* 3. You loaded a protein sample to a SP column. The target protein (pI= 9.5) somehow can not be eluted from the column using pH 7.5 buffer containing 1 M NaCl, what should you do to elute this protein from column (very important: do not use harsh condition to destroy enzymatic activity). This protein will aggregate and lose enzyme activity at 1.1 M NaCl salt concentration and < pH 6 and >pH 10

* 4. Design a AKTA Prime Plus program to purify proteins using linear gradient on a 10 ml Q HP column
(describe chromatographic condition: volume, valve, buffer and concentration, flow rate, fraction)

* 5. You are going to desalt a protein sample eluted from Ni resin on a Sephadex G-25 column (5 X10 cm) , the elution volume is 200 ml. What will you do?

* 6. You need to purify an enzyme (sample from Ni resin, protein concentration is 15 mg/ml, protein pI is 8.5, it will form aggregate when protein concentration is > 16 mg/ml) on a TSKgel G2000SW (21.5X60 cm) column, describe a protocol including a simplified AKTA Prime program, sample prepartion, buffer composition and AKTA operation instruction for such purification.

* 7. What is the optimal imidazole concentration in the lysis buffer for purifying the following proteins?
1.

start
ATG AAT CAC AAA GTG

TCCCCTATACTAGGTTATTG

GAAAATTAAGGGCCTTGTGCAACCCACTCGACTT
CTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGAT
AAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATT
GATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCAC
AACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTT
TTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAA
GTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCAT
AAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTT
GATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTT
AAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATA
GCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGC

GACCATCCTCCAAAATCGGAT
3C
CTGGAAGTTCTGTTCCAGGGCCCGGGA
L E V L F Q G P G
Nde1
catatggc gaccaaaggc accaaacgat cttacgaaca
61 gatggagact gatggagaac gccagaatgc cactgaaatc agagcatctg tcggaaaaat
121 gattgatgga attggacgat tctacatcca aatgtgcacc gaacttaaac tcagtgatta
181 tgagggacgg ctgattcaga acagcttaac aatagagaga atggtgctct ctgcttttga
241 cgagaggagg aataaatatc tagaagaaca tcccagtgcg gggaaagatc ctaagaaaac
301 tggaggacct atatacagga gagtagatgg aaagtggagg agagaactca tcctttatga
361 caaagaagaa ataagacgaa tctggcgcca agctaataat ggtgacgatg caacggctgg
421 tctgactcac atgatgatct ggcactccaa tttgaatgat gcaacttacc agaggacaag
481 agctcttgtt cgcacaggaa tggatcccag gatgtgctca ctgatgcagg gttcaaccct
541 ccctaggagg tctggggccg caggtgctgc agtcaaagga gttggaacaa tggtgatgga
601 attgatcaga atgatcaaac gtgggatcaa tgatcggaac ttctggaggg gtgagaatgg
661 acggagaaca aggattgctt atgaaagaat gtgcaacatt ctcaaaggga aatttcaaac
721 agctgcacaa agaacaatgg tggatcaagt gagagagagc cggaatccag gaaatgctga
781 gttcgaagat ctcatctttt tagcacggtc tgcactcata ttgagagggt cagttgctca
841 caagtcctgc ctgcctgcct gtgtgtatgg atctgccgta gccagtggat acgactttga
901 aagagaggga tactctctag tcggaataga ccctttcaga ctgcttcaaa acagccaagt
961 atacagccta atcagaccaa atgagaatcc agcacacaag agtcaactgg tgtggatggc
1021 atgccattct gctgcatttg aagatctaag agtatcaagc ttcatcagag ggacgaaagt
1081 ggtcccaaga gggaagcttt ccactagagg agttcaaatt gcttccaatg aaaacatgga
1141 gactatggaa tcaagtaccc ttgaactgag aagcagatac tgggccataa ggaccagaag
1201 tggagggaac accaatcaac agagggcttc ctcgggccaa atcagcatac aacctacgtt
1261 ctcagtacag agaaatctcc cttttgacag accaaccatt atggcagcat tcactgggaa
1321 tacagagggg agaacatctg acatgagaac cgaaatcata aggctgatgg aaagtgcaag
1381 accagaagat gtgtctttcc aggggcgggg agtcttcgag ctctcggacg aaaaggcaac
1441 gagcccgatc gtgccctcct ttgacatgag taatgaagga tcttatttct tcggagacaa
1501 tgcagaggag tacgacaatt aa
ctcgag

2.
ATG AGA TTT CCT TCA ATT TTT ACT GCT GTT TTA TTC GCA
GCA TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA
ACG GCA CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA
GGG GAT TTC GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC
GGG TTA TTG TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA
GAA GGG GTA TCT CTC GAG AAA AGG
TGC GCA ATTGTCGGAGGAAGTGACTCCAGAGAAGGAGCCTGGCCTTGGGTCGTTGCTCTGTATTTCGACGATCAACAGGTCTGCGGAGCTTCTCTGGTGAGCAGGGATTGGCTGGTGTCGGCCGCCCACTGCGTGTACGGGAGAAATATGGAGCCGTCTAAGTGGAAAGCAGTGCTAGGCCTGCATATGGCATCAAATCTGACTTCTCCTCAGATAGAAACTAGGTTGATTGACCAAATTGTCATAAAC
Cgt
CACTACAATAAACGGAGAAAGAACAATGACATTGCCATGATGCATCTTGAAATGAAAGTGAACTACACAGATTATATACAGCCTATT
aGc
TTACCAGAAGAAAATCAAGTTTTTCCCCCAGGAAGAATTTGTTCTATTGCTGGCTGGGGGGCACTTATATATCAAGGTTCTACTGCAGACGTACTGCAAGAAGCTGACGTTCCCCTTCTATCAAATGAGAAATGTCAACAACAGATGCCAGAATATAACATTACGGAAAATATGGTGTGTGCAGGCTAT
GAt
GCAGGAGGGGTAGATTCTTGTCAGGGGGATTCAGGCGGACCACTCATGTGCCAAGAAAACAACAGATGGCTCCTGGCTGGCGTGACGTCATTTGGATATCAATGTGCACTGCCTAATCGCCCAGGGGTGTATGCCCGGGTCCCAAGGTTCAC
AGAGTGGATACAAAGTTTTCTACATCATCATCATCATCAcTAG
Kpn1
GGTAC C

* 8. List buffers required for 4-step purification of His-(TEV)-FN3K protein using these columns: HisTrap FF, 5 ml; HiPrep 26/10 Desalting; Q HP RESOURCE Q, 10 ml; HiLoad 16/60 Superdex 200 pg; and Turbo His-TEV protease. Describe chromatographic procedure

* 9. List buffers required and describe chromatographic procedure: Purification and on-column cleavage of His-GST(3c)-FluNA using Turbo His-3C protease and GSTrap FF. Removal of 3C protease using HisTrap FF column in series with GSTrap FF column

* 10. List buffers required and describe chromatographic procedure: Purification and on-column cleavage of His-MBP-(TEV)-FluHA using Turbo His-TEV protease and MBPTrap HP 5 ml. Removal of TEV protease using HisTrap FF column in series with MBPTrap HP column; Polishing using Superdex 200 pg in XK 16/20.
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