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抗體與蛋白A、蛋白G的結(jié)合力：

Note:-means no combination; + means weak combination; ++ means medium combination; +++ means very strong combination.






Application examples:


Binding buffer: 20 mM phosphate buffer (PB), pH 7.4


Elution buffer: 0.1 M glycine-hydrochloric acid buffer, pH 2.7


Neutralization buffer: 1 M Tris-HCl, pH 9.0


1) 1 ml recombinant protein G sepharose gel pre-packed column, wash away the preservation solution with 5-10 column volumes of distilled water;


2) Equilibrate 5-10 bed volumes with binding buffer;


3) Dilute 5 ml of rabbit polyantiserum to 50 ml with binding buffer, filter and load with 0.45 μm filter membrane;


4) Equilibrate 5-10 column bed volumes with binding buffer to baseline;


5) Elute the antibody with elution buffer, collect the elution peak, and adjust its pH to neutral with neutralization buffer;


6) Regenerate and clean with 3 ~ 5 column bed volumes of elution buffer after each use;


7) The purity of the rabbit polyclonal antibody eluted by SDS-PAGE (Figure 1) electrophoresis analysis is above 95%.

圖 1

Precautions:


1) The pre-packed column is easy to use and can complete the purification task without equipment.


2) After the sample is eluted, the pH is generally very low, and the collected antibody solution should be neutralized to neutral with an alkaline neutralization buffer (such as 1 M Tris-HCl, pH 9.0) immediately, or pre-filled in the collection container 5% ~ 20%, pH 7 ~ 9 buffer (such as 1 M Tris-HCl or 1 M phosphate buffer) to help maintain the biological activity of the antibody and avoid antibody inactivation.


3) When using, ensure that the temperature of the column and the buffer are the same to avoid bubbles in the column bed and affect the purification effect. If bubbles have already occurred, you can repack the column by yourself.


4) Normally, after the target sample is eluted, continue to wash 3 to 5 column volumes with 0.1 M glycine-hydrochloric acid buffer (pH 2.0 ~ 2.7), and then wash more than 5 column volumes with equilibration buffer for simple regeneration.


5) In-place cleaning should be performed after several times of use. It is recommended to wash 1 to 3 column volumes (10 to 30 min) with 0.1% Triton X-100 at 37°C, and immediately wash more than 5 column volumes with equilibration buffer; or 70 Wash with% ethanol for more than 5 column volumes (can be maintained for 4-6 h), and then wash with equilibration buffer for more than 5 column volumes. Cleaning-in-place can remove strong hydrophobic proteins and lipids and restore the load and flow rate of the column.