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Phosphoprotein enrichment kit

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
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ºü¸£°í, ƯÀÌÀûÀÎ affinity ±â¹ÝÀÇ phosphoproteinsÀÇ ³óÃà
  • Nondenaturing method·Î ´Ü¹éÁú ±¸Á¶¿Í ¿ëÇؼºÀ» À¯Áö
  • ÃÖÁ¾ ¿ëÃâ¾×¿¡´Â ¼¼Æ÷ Á¾·ù¿¡ µû¶ó¼­ 14-17% phosphoprotein ÇÔÀ¯
  • ºñ¹æ»ç¼º ¼¼Æ÷ ½ÅÈ£ ¿¬±¸ ¹× 2D-PAGE¿¡ ÀûÇÕ
Phosphoprotein Enrichment Kit´Â mammalian ¼¼Æ÷¿Í Á¶Á÷À¸·ÎºÎÅÍ ÀλêÈ­µÈ ´Ü¹éÁúÀ» ºÐ¸®Çϱâ À§ÇÑ È¿°úÀûÀÎ affinity ±â¹ÝÀÇ ¹æ¹ýÀ» Á¦°øÇÑ´Ù (Figure1; 1-2). °¢ Á¦Ç°Àº serine, tyrosine, threonineÀ» Æ÷ÇÔÇÑ º¯ÇüµÈ ¾Æ¹Ì³ë»ê¿¡ »ó°ü¾øÀÌ cytosolÀ̳ª ¸·¿¡ °áÇÕµÈ phosphoproteinÀ» ³óÃàÇϱâ À§ÇÑ 6 °³ÀÇ ³ôÀº ¼ö¿ë·ÂÀ» °®´Â gravity column°ú ÇÔ²² »ç¿ëÇÏ´Â ¿Ïº®ÇÑ buffer ¼¼Æ®¸¦ °¡Áö°í ÀÖ´Ù.
°íµµ·Î ¼±ÅÃÀûÀÎ PhosphoproteinÀÇ ³óÃà
Phosphoprotein Enrichment Kit´Â ¾î¶² mammalian ¼¼Æ÷¿Íµµ ÇÔ²² »ç¿ëÇÒ ¼ö ÀÖ´Ù. °ËÅäµÈ ¼¼Æ÷ Á¾·ù¿¡´Â NIH 3T3, HEK 293, HeLa, Cos-7, JurkatÀÌ ÀÖ´Ù. ³óÃà ÀÛ¾÷Àº Western blotting ºÐ¼®¹ýÀ¸·Î È®ÀÎÇÑ °á°ú ¾ÆÁÖ È¿À²ÀûÀ̾úÀ¸¸ç, (Figure 1) colorimetric phosphate detection¹ýÀ» »ç¿ëÇÏ¿© ´ë´Ù¼öÀÇ phosphoproteinÀÌ ºÐ¸®µÇ¾úÀ½À» È®ÀÎÇÏ¿´°í, wash fraction¿¡¼­´Â ¾ÆÁÖ ±Ø¼Ò·®ÀÇ ´Ü¹éÁúÀ» È®ÀÎÇÏ¿´´Ù.


Figure 1. Highly effective phosphoprotein enrichment. A Phosphoprotein Affinity Column was loaded with ~3 mg of total protein from HEK 293 cells. The extract (Lanes 1), flowthrough (Lanes 2), wash (Lanes 3), and eluate (Lanes 4) were then analyzed by Western blotting using antibodies specific for phosphorylated AKT (Panel A), PTEN (Panel B) & GSK3¥â (Panel C) proteins.
Components & Storage Conditions
°¢ Á¦Ç° ±¸¼º¹°°ú º¸°üÁ¶°ÇÀº Certificate of Analysis ¸¦ ÂüÁ¶ÇϽʽÿÀ.
References
1. BD Phosphoprotein Enrichment Kit (April 2003) Clontechniques XVIII(2):4-5. 2. BD Phosphoprotein Enrichment Kit (July 2004) Clontechniques XIX(3):12-13.

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