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        Antibody Purification

        互联网

        2592
         

        Antibody Purification

        This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinity chromatography.

         

        Solutions

        Affigel Blue Prewash

        0.1 M acetic acid 5.7 ml glacial acetic acid

        1.4 M NaCl 81 g NaCl

        40% isopropanol 400 ml isopropanol

        up to 1 liter with Q

        check to make sure pH is 3.0

        Affigel Blue Running Buffer

        10 mM K2HPO4 2.28 g K2HPO4

        0.15 M NaCl 8.2 g NaCl

        0.02% azide 0.2 g Na azide

        up to 1 liter with Q

        1.4 M NaCl

        8.1 g NaCl

        up to 100 ml with Q

        Saturated NH4SO4

        767 g NH4SO4

        add 1 liter Q

        10X PBS

        80 g NaCl

        2 g KCl

        14.4 g Na2HPO4

        2.4 g KH2PO4

        up to 1 liter with Q

        Affigel Blue Regeneration Buffer

        2 M guanidine HCl or

        1.5 M Na thiocyanate

        HiTrap Storage Buffer

        10 mM Tris 7.5 1 ml 1M Tris 7.5

        0.1 mM EDTA 20 ul 500 mM EDTA

        50 mM NaCl 1 ml 5M NaCl

        0.1% azide 0.1 g Na azide

        up to 100 ml with Q

        1X Coupling Buffer

        0.2 M NaHCO3 1.68 g NaHCO3

        0.5 M NaCl 2.92 g NaCl

        pH to 8.0 and bring up to 100 ml

        1X Buffer A

        0.5 M NaCl 2.92 g NaCl

        0.01 M Tris 0.121 g Tris base

        pH to 8.3 and bring up to 100 ml

        Add 3.0 ml ethanolamine before use

        1X Buffer A

        0.1 M NaOAc 0.82 g NaOAc

        0.5 M NaCl 2.92 g NaCl

        pH to 4.0 and bring up to 100 ml

         

        Procedure

        • Pour a 5 ml affigel blue column (biorad) and wash with 50 ml Prewash to prep the column (when first used or if last used in more than a week).

        • Wash with 50 ml Q, followed by 50 ml Running Buffer.

        • Wash with 50 ml 1.4 M NaCl, if eluate is colored, then re-equilibrate.

        • Wash with 50 ml Running Buffer.

        • Load 1 ml serum, save flow-through, elute with 2 bed volumes running buffer (the serum albumin should stick to the column and the Ig should flow through).

        • On ice slowly add saturated ammonium sulfate to 45% (550 ml sample + 450 ml saturated ammonium sulfate). Tilt overnight at 4°C.

        • Pellet by spinning at 3,000 rpm for 10 minutes at 4°C.

        • Resuspend the pellet in 1 ml 1X PBS on ice (don’t vortex or agitate) and dialyze against PBS.

        • The pharmacia HiTrap 1ml column can bind 10 micromoles of peptide or protein per ml of bed volume. Prep the HiTrap column by washing with 10 ml 50% Isopropanol, 25% Isopropanol, 10% Isopropanol, and 10 ml 1 mM ice cold HCl.

        • Resuspend the peptide or protein in 1 ml 1X Coupling Buffer and load the column. Hold at room temperature for 1 hour.

        • Wash with 5 ml 1X Coupling Buffer and save the flowthrough.

        • Wash with 6 ml Buffer A, 6 ml Buffer B, and 6 ml Buffer A. Hold at room temperature for 30 minutes.

        • Wash with 6 ml Buffer B, 6 ml Buffer A, and 6 ml Buffer B. Wash with Storage Buffer and hold at 4°C.

        • To purify the antibody, load the affigel concentrate onto the column and hold at room temperature for 10 minutes.

        • Wash with 50 ml 10 mM Tris 7.5 and then wash with 50 ml 10 mM Tris 7.5/500 mM NaCl. Elute with 5 ml 100 mM Glycine pH 2.5 into 1 ml 1M Tris 8.0.

        • Dialyze and concentrate with a centricon 30.

        <center> <p>  </p> </center>
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