https://eln.excellgen.com/index.php?action=history&feed=atom&title=Purification_of_scIHFPurification of scIHF - Revision history2026-10-19T21:55:07ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=Purification_of_scIHF&diff=11862&oldid=prevPaul.xu at 16:37, 14 May 20142014-05-14T16:37:34Z<p></p>
<p><b>New page</b></p><div>== Purification of scIHF ==<br />
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<br>1. grow cells in LB to OD600 0.4~ 0.6<br>2. Add 1 mM IPTG, induce for 4 h<br>3. Spin down cells<br>4. Resuspend in Lysis Buffer.<br><br />
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Insoluble fraction: use this buffer: 0.1 M NaH2PO4, 10 mM Tris, 0.1% NP-40, 8 M Urea, pH8.0<br />
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5. Sonicate<br>6. Spin and collect supernatant<br><br />
<pre>Alternative steps: 7 to 11 for non tag protein<br />
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7. Precipitate with 50% - 80% ammonium sulfate.<br />
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8. Resuspend the pellet and dialyze against 100mM NaCl, 25mM HEPES pH 7.0 and 10% glycerol.<br />
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9. Load on a SP Sepharose column. <br />
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10. Wash with 150mM and 250mM NaCl in 25mM HEPES pH 7.0<br />
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11. Elute with 350mM NaCl step in 25mM HEPES pH 7.0, 0.1mM EDTA, and 5% glycerol. <br />
</pre><br />
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7. Load to Ni column.<br>8. Wash with Lysis Buffer<br> <br />
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9. Wash extensively with 50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole pH 8.0 to remove urea. <br />
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10. Elute with Elution Buffer 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0<br>11. Concentrate and then dialysis vs 0.1 M NaH2PO4, 0.1 mM EDTA, 1 mM DTT, 50 mM NaCl, 50% glycerol<br> <br />
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12. Store at -80 oC.<br> <br />
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Amino acid sequence of scIHF<br> <br />
<pre>beta<br />
Nco1<br />
cc atg gcg accaagtcagaattgatagaaagacttgccacccagcaatcgcacattcccgccaag<br />
M A T K S E L I E R L A T Q Q S H I P A K <br />
acggttgaagatgcagtaaaagagatgctggagcatatggcctcgactcttgcgcagggc<br />
T V E D A V K E M L E H M A S T L A Q G <br />
<br />
GGC AGC GGC GGT<br />
G S G G<br />
<br />
alpha<br />
cttacaaaagctgaaatgtcagaatatctgtttgataagcttgggcttagcaag<br />
L T K A E M S E Y L F D K L G L S K <br />
cgggatgccaaagaactggttgaactgtttttcgaagagatccgtcgcgctctggaaaac<br />
R D A K E L V E L F F E E I R R A L E N <br />
ggcgaacaggtgaaactctctggttttggtaacttcgatctgcgtgataagaatcaacgc<br />
G E Q V K L S G F G N F D L R D K N Q R <br />
ccgggacgtaacccgaaaacgggcgaggatattcccattacagcacggcgcgtggtgacc<br />
P G R N P K T G E D I P I T A R R V V T <br />
ttcagacccgggcagaagttaaaaagccgggtcgaaaacgct<br />
F R P G Q K L K S R V E N A <br />
<br />
GGC GGC GGT<br />
G G G<br />
<br />
beta<br />
gagcgtattgaaatccgcggtttcggcagtttctctttgcactaccgcgcaccacgtacc<br />
E R I E I R G F G S F S L H Y R A P R T <br />
ggacgtaatccgaagactggcgataaagtagaactggaaggaaaatacgttcctcacttt<br />
G R N P K T G D K V E L E G K Y V P H F <br />
aaacctggtaaagaactgcgcgatcgcgccaatatttacggt<br />
K P G K E L R D R A N I Y G <br />
Xho1<br />
GGT AGC GGC CAC CAC CAC CAC CAC CAC TGA ctc gag GATCag<br />
G S G<br />
</pre> <br />
[[Category:Protocols]]</div>Paul.xu