Product Name: Alkali-resistant Protein A Sepharose FF


English name: Alkali-tolerant Protein A NUPharose Fast Flow, rat-PA NUPharose FF


Specifications: 20 ml, 100 ml, 1 L, 1 ml pre-packed column, 5 ml pre-packed column, special specifications customized


Transportation: 2 ~ 25℃, normal pressure, avoid light


Storage: 20% ethanol, 2 ~ 8℃


Shelf life: 3 years






Related introduction:


Compared with ordinary protein A agarose gel, Alkali-resistant Protein A Sepharose FF mainly improves the alkali resistance of the gel. It can be washed with 0.1 ~ 0.5 M NaOH solution and remove the heat source. The extended carbon arms and directional binding sites are respectively designed on the bond between the skeleton and the recombinant alkali-resistant protein A to ensure the directional and firm bonding of the recombinant alkali-resistant protein A to the backbone and the high-efficiency affinity with the antibody.






Technical index:


Matrix


4% highly cross-linked agarose gel


Ligand


Recombinant Alkali Tolerant Protein A


Ligand Density


≥6 mg/ml


Filler particle size


60 ~ 180 μm


Maximum flow rate


800 cm/h


Recommended flow rate


20 ~ 100 cm/h


Working (cleaning) pH


3 ~ 11 (2 ~ 13)


Back pressure resistant


0.3 MPa


Capacity


≥40 mg/ml IgG






Features:


1) Ligand shedding: about 1~7 ng rat-PA/mg IgG, the matching rat-PA kit (NRPB53S) detects the residual rat-PA content in the eluate for multiple uses


2) Alkali resistance


Under the condition of 25℃, immerse rat-PA in different concentrations of NaOH solution:


The loading capacity of 0.1 M treatment for 72 h is maintained above 90%;


More than 80% for 0.5 M treatment for 16 h, and more than 50% for 36 h;


1.0 M treatment was more than 90% for 4 h, and more than 50% for 8 h.


Picture 39.jpg






3) Acid resistance


Under the condition of 25℃, immerse rat-PA in acid solutions of different pH:


The capacity of pH 3.0, pH 2.0, 0.1 M HCl (pH 1.0) is maintained above 90% for 6 days


0.5 M HCl treatment is more than 80% for 2 days, and more than 50% for 6 days






Picture 40.jpg






4) Pressure resistance


Instrument: AKTA explorer 100


Pillar: 1.0 cm×30 cm


Flow rate: increase by 0.5 ml/min every 30 s


Picture 41.jpg


5) Service life


The IgG in bovine serum was purified for 200 cycles, and treated with 0.1 M NaOH for 15 minutes each time. No significant load loss was found after 200 uses.


Picture 1.png






Application examples:


Binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.4


Elution buffer: 0.1 M citrate buffer, pH 4.0


Neutralization buffer: 1 M Tris-HCl, pH 9.0


1) 1 ml Alkali-resistant Protein A Sepharose FF pre-packed column is washed with 5-10 column volumes of distilled water to remove the preservation solution;


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


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


4) Wash with binding buffer for another 5-10 bed volumes;


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 0.1 M NaOH solution after each use;


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


Picture 2.png






figure 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. If bubbles have already occurred, you can repack the column by yourself.


4) After use, it should be cleaned in place. Generally, it is recommended to clean 1 to 3 column volumes (10 to 30 min) with 0.1 M NaOH, and immediately wash more than 5 column volumes with equilibration buffer.


5) For in-place cleaning with higher requirements (disinfection, removal of endotoxin, ultra-fast protein residues, etc.), you can first clean with a balance buffer containing a reducing agent (such as 100 mM DTT, β-ME or 1-mercaptoglycerol) 3 ~ 5 column volumes, then wash 1 ~ 5 column volumes (10 ~ 30 min) with 0.1 ~ 0.5 M NaOH, and then wash more than 5 column volumes with sterile endotoxin-free equilibration buffer.


6) Although this product has good alkali resistance, long-term use of higher NaOH concentration (such as 0.5 mol/L) for cleaning will still shorten the service life. It is recommended to clean with 0.1 M NaOH every 1 to 3 times. Wash with 0.5 M NaOH every 5 to 20 times.


7) In order to better protect the packing, strong pH changes should be avoided. It is recommended that after acidic elution of the target antibody, it should be restored to neutrality with equilibration buffer, and then washed with NaOH.


8) This product has a very low ligand shedding rate (<10 ng/mg IgG), combined with alkali resistance, and is especially suitable for the production of monoclonal antibodies. However, experiments have found that the first use of the ligand after long-term storage will have a higher shedding rate. It is recommended that for the first use after long-term storage, first wash 3 to 5 column volumes with an acidic eluent, and then equilibrate and purify the sample with an equilibration buffer.


Recommended flow rate


Prepacked column volume 1 ml 5 ml 10 ml


Flow rate (ml /min) 0.2 1 2


9)










Recommended flow rate


Prepacked column volume


1 ml


5 ml


10 ml


Flow rate (ml /min)


0.2


1


2

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