Protein A+G Agarose (Fast Flow, 进口分装)

Item Number

P749481

• Product Name: Protein A+G Agarose (Fast Flow, 进口分装)

Product Description

Protein A+G Agarose is mainly used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and antibody purification.Protein A+G Agarose is suitable for immunoprecipitation of all antibodies that Protein A Agarose and Protein G Agarose alone can immunoprecipitate, including mouse IgG1, IgG2a, IgG2b, IgG3, IgA, rat IgG1, IgG2a, IgG2b, IgG2c, rabbit IgG, rabbit and goat polyclonal Abs, as well as human IgG1, IgG2, IgG3 and IgG4.Both Protein A and Protein G are covalently crosslinked to 4% beaded agarose . Two milliliters of Protein A+G Agarose contains a total of approximately 2 mg of recombinant Protein A and Protein G, with a binding capacity of approximately 15mg human IgG. The recommended linear flow rate is 50-300 cm/h.This product is formulated in TBS solution, supplied as a 25% slurry (e.g., 0.5ml of settled resin is equivalent to 2ml of 25% slurry).This product is sufficient for 100 immunoprecipitations.

Precautions：

Do not freeze this product.This product must be fully resuspended before use by inverting the tube several times.Protein samples should always be placed at 4℃ or on ice to minimize protein degradation or denaturation.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. Immunoprecipitation a. Preparation of protein samples:(a) For adherent cells in 10cm cell culture dishes, aspirate the culture medium, wash once with PBS, then lyse cells with 0.5-2ml of cell lysis buffer. We recommend using 's Cell Lysis Buffer for Western and IP or RIPA lysis buffers .(b) For tissue samples, directly lyse cells after homogenization.(c) For suspension cells, collect cells by centrifugation, wash them once with PBS, then lyse cells as described for adherent cells.Note: Lysis of cell or tissue samples should be performed according to the instructions for different lysis buffers. If the protein concentration obtained is too high, the lysate can be diluted appropriately with lysis buffer or PBS. In case of too low protein concentration, the amount of lysis buffer used for sample lysis should be reduced appropriately.b. (Optional) Removal of non-specific binding:(a) Take 0.2-1ml of protein sample (0.2-1mg total protein), add approximately 1µg of IgG of the same species as the IgG used for immunoprecipitation and 20µl of well resuspended Protein A+G Agarose. Shake slowly at 4℃ for 30 minutes to 2 hours.Note: For example, if mouse IgG is used for in immunoprecipitation, normal mouse IgG should be added in this step. By incubating with normal IgG and Protein A+G Agarose, the background due to non-specific binding can be significantly reduced.(b) Centrifuge at 2500rpm (~1000×g) for 5 minutes and take the supernatant for immunoprecipitation subsequently.c. Immunoprecipitation:(a) Add 0.2-2µg of primary antibody into the supernatant obtained in the previous step, and shake slowly overnight at 4℃.(b) Add 20µl of fully resuspended Protein A+G Agarose and shake slowly at 4℃ for 1-3 hours. Note: The amount of Agarose can be adjusted to 40µl to facilitate washing subsequently.(c) Centrifuge at 2500 rpm (approximately 1000×g) for 5 minutes or have a quick spin at high speed, and carefully aspirate the supernatant.(d) Wash the settled Protein A+G Agarose 5 times with 0.5-1ml of lysis buffer or PBS used for protein sample preparation. After each wash, centrifuge at 2500 rpm (approximately 1000×g) for 5 minutes or have a quick spin at high speed to remove the wash solution.(e) After the last wash, remove the supernatant and resuspend the Protein A+G Agarose with 20-40µl of 1X SDS-PAGE loading buffer by vortex or pipetting. Pulse-spin to collect droplet.(f) Boil at 100℃ for 3-5 minutes and take some or all of the samples for SDS-PAGE analysis. The sample can be stored at -20℃ for later analysis.2. Co-immunoprecipitation: Refer to a protocol for Co-immunoprecipitation. Fresh, unfrozen protein samples are strongly recommended for Co-IP assays. For general immunoprecipitation, fresh protein samples are also preferred, although frozen protein samples can be used.3. Antibody purificationa. Preparation:(a) Filter all solutions used with 0.45µm or 0.2µm pore size filter membranes.(b) All solutions must be degassed by methods such as ultrasound.(c) Select an empty purification column and load an appropriate amount of Protein A+G Agarose.(d) Equilibrate the column with 10-20 times the bed volume of TBS at a flow rate of 1 ml/min controlled by a constant flow pump. If a constant flow pump is not available, the column can also be washed and equilibrated entirely by gravity.b. Antibody purification:(a) Load the protein sample containing the antibody onto the purification column and allow it to flow completely into the resin. Do not allow the resin bed to run dry.(b) Wash the Protein A+G column with 10-20 times the bed volume of TBS to remove all non-bound and non-specifically bound proteins. Whether the wash is complete can be determined by measuring the absorbance of the flow through at 280 nm. The last flow through fractions should have absorbance similar to TBS. (c) Elute antibodies with 10ml of Elution Buffer (50 mM glycine, pH 2.7). Some antibodies that bind strongly to Protein A+G and are hard to elute at pH2.7 can be eluted with 50 mM glycine at pH 1.9. Collect the eluted antibodies in separate tubes and determine in which tubes the elution peaks are located by measuring the absorbance at 280nm or by protein assays.c. Regeneration of purification columns:(a) Wash the purification column with 10-20 times the bed volume of TBS to bring the purification column to a neutral pH.(b) Use TBS to preserve the regenerated purification column.Related Products:产品编号产品名称包装P2006Protein A Agarose 2mlP2009Protein G Agarose 2mlP2012Protein A+G Agarose 2ml