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Purification of chromosomal ParA1 protein for raising polyclonal antibodies

Purification of chromosomal ParA1 protein for raising polyclonal antibodies. 王群中. Chun-Chung Wang Department of Life Sciences National Normal University. What’s ParA1 ?. pSOT29. Purification scheme. Step 1 :prepare BL21(DE3)/pSOT29 、 BL21(DE3)/pLysS/pSOT29

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Purification of chromosomal ParA1 protein for raising polyclonal antibodies

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  1. Purification of chromosomal ParA1 protein for raising polyclonal antibodies 王群中 Chun-Chung Wang Department of Life Sciences National Normal University

  2. What’s ParA1 ?

  3. pSOT29

  4. Purification scheme Step 1 :prepare BL21(DE3)/pSOT29、BL21(DE3)/pLysS/pSOT29 Step 2: Use IPTG to induce cell to produce protein Step 3: Affinity chromatography

  5. 1 2 3 4 5 Result: testing induction conditions KDa 250 150 100 75 50 Trx-His-ParA1 37 25 1、Marker 20 2、before induction : pellet 15 3、before induction : supernatant 4、after induction : pellet 5、after induction : supernatant

  6. THE END

  7. BL21/pLysS/pSOT29 KDa 250 150 100 75 1、Marker 50 Trx-His-ParA1 37 2、before induction : pellet 3、before induction : supernatant 25 20 4、after induction : pellet 5、after induction : supernatant 1 2 3 4 5

  8. www1.nttu.edu.tw/yenlee/new_page_15.htm • http://journals.iucr.org/e/issues/2005/08/00/ng6161/ng6161bdy.html • Novagen

  9. Lac operon Lactose

  10. Lac operon IPTG

  11. Step 3 Break cell Affinity chromatography

  12. Affinity chromatography principle • Affinity column can bind to protein • And wash off other cellular components • Then elute the target protein

  13. How does His-tag bind to protein? Binding protein

  14. How to elute protein?

  15. step1 • plasmid isolation from MC1000/pSOT29 • Transformation with BL21 or BL21/pLysS • Get the BL21/pSOT29 or

  16. Use IPTG to induce cell to produce protein step2 IPTG

  17. pLysS’s Function • pLysS contains a T7 lysozyme gene • T7 lysozyme can inhibit T7 polymerase • IPTG can induce T7 polymerase gene present greater than T7 lysozyme can inhibit

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