Affinity layering purifys polyclonal antibodies.

The experimental principle the high selectivity of affinity and layering makes it possible to purify from an initial material and to collect a lower content of the purpose protein, so affinity layering is one of the most effective methods in the separation and purification process of
protein
. In addition, if the base and protein affinity is very strong, samples can also be concentrated at the same time.although in most cases it is not necessary to separate the
antibody
from other
serum
proteins, protein A affinity is a very effective separation method if needed. Protein A is obtained from Staphylococcus aureus and can be combined with Fc fragments of the weight chain. Protein A is now known to be combined with IgG in a variety of mammals or with certain IgM and IgA.
If protein A is connected to a solid-phase vector, such as the sepharose CL-4B, this filler can be an important tool for separating and purifying different types of different subsectors or antibody pieces.: experimental materials. Experimental instruments
protein A column (containing 10 ml or 5 ml filler); pump; centrifuge tube;
centrifuge
;p H test paper;
filtration
; glass column.
2. Experimental
Reagent (1) TBS buffer solution: 6.06g Tris (50mM), 8.78g NaCl (150mM) and 0.5g sodium nitride (0.05%) dissolved in 1L distilled water and regulated pH 7.4 with HCl.
(2) medium buffer solution: 121.2g Tris (1M), 87.8g NaCl (1.5M), 0.37g
. EDTA
(1mM) and 5g of sodium nitride (0.5%) dissolved in 1L distilled water and adjusted with HCl pH8.0.
(3) wash-out buffer solution (pH2.7): Dissolve 3.75g glycine (50 mM) in 1L distilled water and adjust pH 2.7 with HCl.
"Experimental Methods" . 1. Prepare the protein A sepharose CL-4B affinity column
Usually prepare 5 ml or 10 ml protein A sepharose CL-4B filler, mix the equal volume filler with the TBS buffer solution in a vacuum bottle and stir. Vacuum for about 15 minutes to remove bubbles from the filler, and bubbles formed in the column on the no side affect the capacity and separation effect of the column.
Slowly adding the protein A sepharose CL-4B filler to the glass column, using the pump control fill speed of 1 ml/min-2 ml/min, avoiding column drying, balancing the column with a TBS buffer solution 10 times the size of the bed and with a pre-cooled TBS buffer solution.
2. Prepare anti-serum
and slowly thaw it in ice water or in a refrigerator at 4 degrees C to avoid protein aggregation. The aggregation that occurs during protein thawing can be dissolved by preheating at 37 degrees C. Add solid laminated sodium nitride to a concentration of 0.05%, 4 degrees C, 15,000xg centrifugation for 5 minutes, remove the clarified anti-serum and filter to remove excess fat.3. Affinity
dilutes the antibody in a 1:5 ratio with a TBS buffer solution and filters it with a filter. At a rate of 0.5 ml per minute, the anti-serum is added to the column, and in order to ensure the combination of anti-serum and filler, the upper column 2 times in a row is required and the upper sample outflow is retained. Wash the post with TBS buffer solution to A<280nm<0.008 and add pH2.7 to wash off the buffer solution at a speed of 0.5 ml/min until all proteins flow down.
Collect the washesis with a 1.5 ml EP tube tube that has been added to the 100ul medium buffer solution, and check the pH of the sequestration with pH test paper after mixing, if the pH is lower than 7 usable medium buffer to about pH7.4 to prevent antibody denaturation.
add 10 ml, pH1.9 wash-out buffer solution to the column, collect the washeated solution according to the above method to A<280nm<0.008.
the protein content in each tube is determined using a terra photonometer. If the protein concentration is less than 0.5 mg/ml can be added to 10% glycerin for preservation, the purified antibody is packed and stored at 2 to 8 degrees C.
the column with a TBS buffer solution containing 0.05% sodium nitride and store the column in an environment of 2 to 8 degrees C. :
SDS
/PAGE to check the purity of the protein obtained by the ealms and the titration of the purified antibody using immunoelectring technology.
note is not . 1. Stacking sodium nitride is toxic, wear gloves and operate carefully
During purification, pre-cooled TBS buffer solution reduces nonse specific binding of proteins and microbial metabolism.
(4) wash-out buffer solution (pH1.9): Dissolve 3.75g glycine (50 mM) in 1L distilled water and adjust pH 1.9 with HCl..