Dynabeads Troubleshooting giude


 

1. Protein A/G beads , IP

2. M-280Tosylactivated beads


 

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Q&A

Tech Tip

Protein A/G beads, IP


Q. Dynabeads¸¦ ÀÌ¿ëÇؼ­ IP½ÇÇèÀ» ¼öÇàÇß½À´Ï´Ù. ±×·±µ¥ cross-linking½ÃŲ ÈÄ IP½ÇÇèÀ» Çغ¸´Ï gel ¾Æ¹«·± bandµµ È®ÀÎÀÌ µÇÁö ¾Ê¾Ò½À´Ï´Ù.

A.

 

1) cross-linking°úÁ¤¿¡¼­ antibodyÀÇ binding site°¡ ¹Ù²î¾úÀ» °¡´É¼ºÀÌ ÀÖ½À´Ï´Ù. ÀÌ·² °æ¿ì antibodyÀÇ affinity°¡ ÁÙ¾îµé°Å³ª affinity¸¦ º¸ÀÌÁö ¾Ê´Â Çö»óÀÌ ¹ß»ýÇÒ ¼ö ÀÖ½À´Ï´Ù. ¶ÇÇÑ cross-linkingÀ» ÅëÇØ non-specific target¿¡ ´ëÇÑ affinity°¡ ¹ß»ýÇÒ ¼öµµ ÀÖ½À´Ï´Ù. ÀÌ·¯ÇÑ Çö»óÀº cross-linkingÀÇ °¡Àå Å« ´ÜÁ¡À̸ç, ´Ù¸¥ covalent antibody couplingÀ» ÅëÇؼ­ ÇØ°áÇÒ ¼ö ÀÖ½À´Ï´Ù.

2) Dynabeads Antibody coupling kitÀ» ÀÌ¿ëÇϼ¼¿ä. ÀÌ kitÀº Dynabeads¿Í antibodyÀÇ covalent coupling½ÃÄÑÁÖ´Â solutionÀÔ´Ï´Ù. Antibody coupling kitÀº °ÅÀÇ ¸ðµç antibody¿¡ ÀûÇÕÇÕ´Ï´Ù. ¶ÇÇÑ ÀÌ kitÀº Dynabeads¿Í antibodyÀÇ covalent couplingÀ» À§ÇØ ¸¸µé¾îÁø kitÀÔ´Ï´Ù. ÀÌ kitÀº cross-linking°ú´Â ´Ù¸£°Ô antibodyÀÇ specificity³ª affinity¿¡ ¿µÇâÀ» ÁÖÁö ¾Ê½À´Ï´Ù.

 

Q. Dynabeads¿¡ antibody¸¦ cross-linkingÇßÁö¸¸ ¿©ÀüÈ÷ elution½Ã¿¡ antibody°¡ ÇÔ²² ³ª¿É´Ï´Ù.

A. ¸ðµç antibody¿¡ ´ëÇؼ­ 100% cross-linkingµÇÁö´Â ¾Ê½À´Ï´Ù. ¸î¸îÀÇ antibodyµéÀº crosslinkingµÇÁö ¾Ê¾Æ¼­ elution µÉ ¼ö ÀÕ½À´Ï´Ù. Cross-linkingÈÄ¿¡ ³·Àº pH¸¦ ÀÌ¿ëÇؼ­ washingÇϸé non cross-linkingµÈ antibody¸¦ Á¦°ÅÇÒ ¼ö ÀÖ½À´Ï´Ù. Wash ÈÄ pH¸¦ ¿ø·¡´ë·Î µ¹·Á³õÀº µÚ IP¸¦ ¼öÇàÇϼ¼¿ä.

 

Q. IP¸¦ ¼öÇàÇϱâ Àü¿¡ non cross-linking antibody¸¦ Á¦°ÅÇßÁö¸¸ ¿©ÀüÈ÷ gel¿¡¼­ antibody°¡ º¸ÀÔ´Ï´Ù.

A. ¸¸¾à gel loadingÀü¿¡ reducing agent¸¦ »ç¿ëÇϼ̴ٸé reducing agent¸¦ »« sample buffer¸¦ »ç¿ëÇغ¸¼¼¿ä. DTT³ª beta-mercaptoethanol°ú °°Àº reducing reagentµéÀº antibody³»ÀÇ disulfide bridge¸¦ ²÷¾î¼­ antibodyÀÇ light chain°ú heavy chainÀ» release½Ãŵ´Ï´Ù. ¶ÇÇÑ ³·Àº pH¸¦ ÀÌ¿ëÇؼ­ proteinÀ» elutionÇϸé antibody¿Í beadÀÇ bindingÀÌ À¯ÁöµÉ °ÍÀÔ´Ï´Ù.

 

Q. IP ½ÇÇè¿¡¼­ non-specific bindingÀÌ º¸ÀÔ´Ï´Ù.

A. Á¶±Ý ´õ °­ÇÑ washing buffer¸¦ »ç¿ëÇغ¸¼¼¿ä. Washing buffer¿¡ non-ionic detergent (tween-20 or triton X-100)À» 0.01-01»çÀÌÀÇ ³óµµ·Î ³ÖÀ¸¸é µË´Ï´Ù. ¸¸¾à beads°¡ ħ°­ Àü¿¡ blockµÆ´Ù¸é washing buffer¿¡ ƯÁ¤ blocker¸¦ ³Ö¾îÁÖ¼¼¿ä. ¶ÇÇÑ washing Ƚ¼ö¸¦ ´Ã¸®°Å³ª washing stepÀ» ´Ã¸®°Å³ª beads¿Í sampleÀÇ incubation ½Ã°£À» ÁÙÀ̰ųª antibodyÀÇ ³óµµ¸¦ ÁÙÀÌ´Â °Íµµ ÇÑ ¹æ¹ýÀÔ´Ï´Ù. ¾Æ´Ï¸é indirect ¹æ¹ýÀ» ½ÃµµÇغ¸¼¼¿ä. Pre-clearing step ¶ÇÇÑ non-specifically bind¸¦ ÁÙ¿©ÁÙ ¼ö ÀÖ½À´Ï´Ù.

 

Q. Dynabeads G¸¦ ±¸¸ÅÇß½À´Ï´Ù. Beads washingÀ» À§ÇØ beads¿Í PBS¸¦ ¼¯¾ú´Âµ¥ PBS »öÀÌ ºÓÀº»öÀ¸·Î º¯Çϸ鼭 beads¿Í magnet »çÀÌÀÇ ºÎÂø·ÂÀÌ ¾àÇØÁý´Ï´Ù.

A. Non-ionic detergent (Tween-20, Triton X-100, surfactant)¸¦ ÃÖÁ¾ ³óµµ 0.01-0.1%°¡ µÇµµ·Ï ³Ö¾îÁØ ÈÄ »ó¿Â¿¡¼­ 5-10ºÐ Á¤µµ incubation ½Ãŵ´Ï´Ù.

 


M-280 Tosylactivated beads


Q. HydrophobicÇÑ molecule°ú M-280 Tosylactivated beadsÀÇ coupling ½ÇÇèÀ» Çϴµ¥ ÀÚ²Ù beads°¡ ²öÀûÇØÁ®¼­ ´õ ÀÌ»ó ½ÇÇè ÁøÇàÀÌ ºÒ°¡´ÉÇÕ´Ï´Ù.

A. M-280 Tosylactivated beads¸¦ ÀÌ¿ëÇÑ coupling½ÇÇè¿¡´Â ÃÑ ¼¼ °³ÀÇ buffer°¡ ÇÊ¿äÇѵ¥¿ä, ÀÌ ¶§ buffer C¿¡´Â ammonium sulphate°¡ Æ÷ÇԵǾî ÀÖ½À´Ï´Ù. ¸¸ÀÏ coupling moleculeÀÇ storage buffer°¡ ³Ê¹« hydrophobicÇÒ °æ¿ì ÀÌ ammonium sulphate¿Í ¹ÝÀÀÇÒ ¼ö Àִµ¥¿ä. ÀÌ ¶§ ammonium sulphateÀÇ ³óµµ¸¦ ÁÙ¿©¼­ ½ÇÇèÇÏ½Ã¸é µË´Ï´Ù.


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