Dynabeads Tech Tip


 

1. Protein A/G beads ¹× IP, Co-IP

 

2. Streptavidin beads


 

Streptavidin beads home

 

Additional Information

Q&A

Protein A/G beads ¹× IP, Co-IP


Q. AntibodyÀÇ °áÇÕ·ÂÀÌ ¾àÇÑ °Í °°½À´Ï´Ù. ¾î¶»°Ô ÇÏ¸é °áÇÕ·ÂÀ» °­È­½Ãų ¼ö ÀÖ³ª¿ä?

 

A. ¸ÕÀú ELISA¿Í °°Àº ¹æ¹ýÀ» ÅëÇؼ­ antibodyÀÇ binding/specificity¸¦ È®ÀÎÇϼ¼¿ä. ±×·± ´ÙÀ½ antibody¿Í beads°¡ bindingÇÏ´ÂÁö¸¦ È®ÀÎÇϼ¼¿ä. ¸¸¾à antibody¿Í beads°¡ bindingÇÏÁö ¾ÊÀ¸¸é IP½ÇÇèÀ» ÁøÇàÇÒ ¼ö ¾ø½À´Ï´Ù. ±×¸®°í beadsÀÇ ¾ç°ú sample volumeÀ» È®ÀÎÇϼ¼¿ä. ÇØ´ç Á¦Ç°ÀÇ insert¿¡¼­ capacity¸¦ È®ÀÎÇÒ ¼ö ÀÖ½À´Ï´Ù. ÀÌ ¶§ beadsÀÇ ¾çÀ̳ª antibodyÀÇ ³óµµ¸¦ ´Ã·Áº¸´Â °Íµµ ÇÑ ¹æ¹ýÀÔ´Ï´Ù. ¶ÇÇÑ incubation ½Ã°£À» ´Ã¸®°Å³ª, ´Ù¸¥ antibody¸¦ »ç¿ëÇغ¸¼¼¿ä.

 

 

Q. Dynabeads protein G ¶Ç´Â A°¡ BSA·Î pre-blocking µÇ¾îÀÖ½À´Ï±î?

 

A. ¾Æ´Ï¿ä. Pre-blocking µÇ¾îÀÖÁö ¾Ê½À´Ï´Ù.

 

 

Q. Dynabeads protein G ¶Ç´Â A¸¦ BSA·Î blocking ÇÏ°í ½Í¾î¿ä.

 

A. BSA´Â hydrophobicÇÑ ¼ºÁúÀ» °¡Áö°í ÀÖ°í Dynabeads protein A/G´Â hydrophilicÇÑ ¼ºÁúÀ» Áö´Ï°í ÀÖ½À´Ï´Ù. ¶§¹®¿¡ BSA·Î pre-blockingÀ» ÇÒ ÇÊ¿ä°¡ ¾ø½À´Ï´Ù. ´Ù¸¸, non-specific bindingÀ» ÁÙÀ̱â À§Çؼ­ washing buffer¿¡ Tween-20 detergent (0.01-0.1%)¸¦ ÷°¡Çؼ­ »ç¿ëÇϽñ⸦ ±ÇÀåÇÕ´Ï´Ù.

 

 

Q. Negative controló·³ Dynabeads protein G¿¡ ¾Æ¹«·± antibody¸¦ ºÎÂøÇÏÁö ¾Ê°í sample°ú incubation ½ÃÄ×½À´Ï´Ù. ÀÌ ¶§ non-specific bindingÀÌ ³ª¿Ô½À´Ï´Ù.

 

A. ¾Æ¹«·± antibody¸¦ ºÎÂøÇÏÁö ¾ÊÀº beads¿Í sampleÀ» ÇÔ²² incubationÇÏ´Â °ÍÀº ÁÁÀº controlÀÇ ¿¹°¡ ¾Æ´Õ´Ï´Ù. Sample³»ÀÇ ´Ù¸¥ ¹°ÁúµéÀÌ protein G³ª beads¿¡ bindingÇÒ ¼ö Àֱ⠶§¹®ÀÔ´Ï´Ù. (hydrophobic interactionÀ̳ª charge interactionµîÀ» ÅëÇؼ­) °¡Àå ÁÁÀº negative controlÀº Dyanbeads protein G¿Í ½ÇÇè°ú ÀüÇô »ó°ü ¾ø´Â IgG¸¦ binding½ÃÅ°´Â °Í ÀÔ´Ï´Ù. ¸¸¾à Á¤¸»·Î ³·Àº levelÀÇ background bindingÀ» ¿øÇÑ´Ù¸é, Dynabeads antibody coupling kitÀ» »ç¿ëÇϼ¼¿ä. ÀÌ kit¿¡¼­ Á¦°øµÇ´Â beads´Â ±²ÀåÈ÷ ÀûÀº background bindingÀ» º¸ÀÔ´Ï´Ù. °¡Áö°í ÀÖ´Â antibody¿Í beads°¡ covalent copingµÇ¸é, elution°úÁ¤¿¡¼­ beads¿Í antibody°¡ ¶³¾îÁöÁö ¾ÊÀ» °ÍÀÔ´Ï´Ù.

 

 


Streptavidin beads


Q. IP¸¦ ¼öÇàÇϱâ À§Çؼ­ Biotinylated antibody¿Í streptavidin beads¸¦ binding½ÃÄ×½À´Ï´Ù. ¾î¶»°Ô antibodyÀÇ elution¾øÀÌ target proteinÀ» elutionÇϳª¿ä?

 

A. High salt (>1M salt) ¶Ç´Â low pH(0.1M glycine-HCL, pH2.5-3.0)ÀÇ bufferµî°ú °°Àº mild elution conditionÀ» ¸¸µì´Ï´Ù. ¶Ç´Â beads¸¦ SDS buffer¿¡ ³ÖÀº ä·Î heat inactivation (95¡É, 5min)À» ÇÏÁö ¸¶¼¼¿ä

 

 

Q. Biotinylated moleculeÀÇ immobilizing¿¡´Â ¾î¶² buffer¸¦ »ç¿ëÇÏ´Â °Ô ÁÁÀº°¡¿ä?

 

A. Biotinylated proteinÀ̳ª ´Ù¸¥ moleculeÀº PBS¸¦, biotinylated nucleic acid´Â B&W buffer¸¦ ÃßõÇص帳´Ï´Ù

*B&W buffer: 10.0 mM Tris-HCL (pH7.5), 1.0 mM EDTA, 2.0 M NaCl

 

 

Q. Streptavidin beads¸¦ Á÷Á¢ÀûÀ¸·Î PCR¿¡ ÀÌ¿ëÇÒ ¼ö ÀÖ³ª¿ä?

 

A. ³×, beads°¡ enzyme reaction¿¡ ¿µÇâÀ» ÁÖÁö ¾Ê±â ¶§¹®¿¡ °¡´ÉÇÕ´Ï´Ù. M-270, MyOne C1, MyOne T1Àº ÀüÇô ¿µÇâÀ» ¹ÌÄ¡Áö ¾Ê½À´Ï´Ù. ´Ù¸¸ M-280 Streptavidin beadsÀÇ °æ¿ì¿¡´Â Àüü 50 ul PCR reaction Áß 75-100ugÀÏ ¶§, ±×¸®°í ³óµµ°¡ 100 ug/50 ul reactionÀ» ³ÑÀ» ¶§ PCR¿¡ ¿µÇâÀ» ÁÖ´Â °ÍÀ» È®ÀÎÇß½À´Ï´Ù.

 

 

Q. Beads·Î biotinylated dsDNA¸¦ ºÐ¸®Çߴµ¥¿ä, biotin ¾øÀÌ dsDNA¸¸ ºÐ¸®ÇÒ ¼ö ÀÖ³ª¿ä?

 

A. BiotinÀ» Á¦°ÅÇÏ´Â ¹æ¹ýÀº µÎ °¡Áö°¡ ÀÖ½À´Ï´Ù.

 

1) Using heat:

 

1. DNA¿Í bindingÇÑ Dynabeads 50ul¸¦ 1x SSC buffer·Î washing

2. Beads¸¦ ¶Ç ´Ù¸¥ 1x SSC buffer 50 ¥ìl·Î resuspension

3. 95 °C¿¡¼­ 5ºÐ µ¿¾È incubation.

4. Magnet¿¡ 1-2ºÐ µ¿¾È ¹æÄ¡ÇÑ ÈÄ »óÃþ¾×À» »õ·Î¿î tube¿¡ ¿Å±è. »óÃþ¾×¿¡ non-biotinylated DNA°¡ Æ÷ÇԵǾî ÀÖ½À´Ï´Ù.

 

2) Using NaOH:

 

1. DNA¿Í bindingÇÑ Dynabeads 50ul¸¦ 1x SSC buffer·Î washing

2. Beads¸¦ 0.15M NaOH 20ul¿¡ resuspension.

3. RT¿¡¼­ 10ºÐ °£ incubation

4. Magnet¿¡ 1-2ºÐ µ¿¾È ¹æÄ¡ÇÑ ÈÄ »óÃþ¾×À» »õ·Î¿î tube¿¡ ¿Å±è. »óÃþ¾×¿¡ non-biotinylated DNA°¡ Æ÷ÇԵǾî ÀÖ½À´Ï´Ù.

5. 2.2ul 10x TE(pH 7.5), 1.3ul 1.25M acetic acid¸¦ »óÃþ¾×¿¡ ³Ö¾î¼­ neutralization. DNA¿Í bindingÇÑ Dynabeads¸¦ ÇÑ ¹øÀº 50ul 0.1M NaOH·Î, ¶Ç ÇÑ ¹øÀº 50ul B&W buffer·Î, ¸¶Áö¸·À¸·Î 50ul TE buffer·Î washing.

*1 x SSC (0.15 M NaCl, 0.015 M sodium citrate. Dissolve the reagents in 800 ml water. Adjust pH to 7.0 with NaOH. Adjust the volume to 1 liter with water).

 

 

Q. Streptavidin-coupled Dynabeads¿Í biotinylated moleculeÀ» ¾î¶»°Ô ºÐ¸®Çϳª¿ä?

 

A. Streptavidin-biotin »çÀÌÀÇ °áÇÕ·ÂÀº ±²ÀåÈ÷ °­ÇÏ´Ù°í ¾Ë·ÁÁ® ÀÖ½À´Ï´Ù. ¶§¹®¿¡ °­·ÂÇÑ ¹æ¹ýÀ» ÅëÇؼ­¸¸ ºÐ¸®ÇÒ ¼ö Àִµ¥¿ä. ÀÚ¼¼ÇÑ Elution guide´Â ¾Æ·¡¸¦ Âü°íÇØÁÖ¼¼¿ä.

 

1) Biotinylated nucleic acids

: 95% formamide+10mM EDTA, pH 8.2 ¿ë¾×¿¡ 65¡É 5ºÐ, ¶Ç´Â 90¡É¿¡¼­ 2ºÐ°£ incubation.

Beads¸¦ magnet¿¡ °íÁ¤½ÃŲ ÈÄ »óÃþ¾×¸¸ ´Ù¸¥ tube¿¡ ¿Å±è. »óÃþ¾×¿¡ biotinylated nucleic acid°¡ µé¾îÀÖÀ½.

 

2) Biotinylated proteins

: 0.1% SDS ¶Ç´Â SDS-PHAGE buffer¸¦ ³Ö°í 3ºÐ°£ ²úÀÓ

 

 

Q. M-280 Streptavidin beads¸¦ ÀÌ¿ëÇؼ­ DNA binding proteinÀ» ÀâÀº ÈÄ oligo¸¦ sample¿¡ ³Ö¾ú½À´Ï´Ù. (Indirect methods). Oligo¿Í beadsÀÇ bindingÀ» À§Çؼ­ B&W buffer¸¦ »ç¿ëÇ϶ó°í µÇ¾î ÀÖ´Â µ¥¿ä, B&W buffer°¡ DNA-protein interaction¿¡ ¿µÇâÀ» ÁÖÁö´Â ¾Ê³ª¿ä?

 

A. B&W buffer´Â proteinÀÌ ¾øÀ» ¶§ oligo¸¦ beads¿¡ °íÁ¤½ÃÅ°´Â direct methods¿¡¼­ »ç¿ëµÇ´Â bufferÀÔ´Ï´Ù. Indirect methods¿¡¼­´Â 150mM ÀÌÇÏÀÇ salt³óµµ¸¦ °¡Áö´Â buffer¸¦ ÀÌ¿ëÇϽñ⸦ ±ÇÀåÇÕ´Ï´Ù.

 

Q. ÀÌ¹Ì BSA·Î coatingµÇ¾î ÀÖ´Â beads¸¦ »ç¿ë ÁßÀÔ´Ï´Ù (MyOne T1, M-280) ±×·±µ¥ °è¼ÓÇؼ­ non-specific binding Çö»óÀÌ º¸À̴µ¥¿ä. Ãß°¡ÀûÀÎ blockingÀ» ¼öÇà Çصµ ±¦ÂúÀº°¡¿ä?

 

A. ³×. Ãß°¡ÀûÀÎ blocking ¼öÇàÀÌ °¡´É ÇÕ´Ï´Ù.

beads¸¦ ÀÌ¿ëÇØ º»°ÝÀûÀÎ ½ÇÇèÀ» Çϱ⿡ ¾Õ¼­, 0.2~0.5% BSA in PBS¸¦ ÀÌ¿ëÇؼ­ 30ºÐ µ¿¾È »ó¿Â¿¡¼­ rotation ½ÃÅ°¸é¼­ blocking ¼öÇàÇÏ½Ã¸é µË´Ï´Ù.

 

 


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