| Product Description | Protein G Resin is mainly used for the purification of antibodies and can also be used for immunoprecipitation (IP) or immunoprecipitation (Co-IP).For IP or Co-IP, Aladdin's Protein G Agarose or Protein G Agarose can also be used.Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42kDa. Protein G is an immunoglobulin binding protein expressed by Streptococcal bacteria type C or G. Protein A and Protein G are functionally similar and bind specifically to mammalian immunoglobulin (Ig), usually to the Fc region of the immunoglobulin, but it has been reported that Protein A also binds to the Fab region of the human VH3 family, and Protein G sometimes binds to the Fab region as well. At the same time, Protein A and Protein G differ in their ability to bind to different immunoglobulin subclasses. Recombinant Protein A and G with appropriate modifications bound to agarose gel can be used for immunoprecipitation or antibody purification.Protein G agarose is suitable for immunoprecipitation of human IgG1, IgG2, IgG3, IgG4, mouse IgG1, IgG2a, IgG2b, IgG3, rat IgG1, IgG2a, IgG2b, IgG2c, as well as rabbit and goat polyclonal antibodies. The following table shows the binding capacity of Aladdin's Protein A, Protein G and Protein A+G agarose products to common immunoglobulin subclasses of human, mouse and rat and the total binding capacity of different species. The recombinant Protein G in this product binds specifically to the Fc region of most mammalian IgGs and has a molecular weight of approximately 22 kDa. The recombinant Protein G has been modified to reduce non-specific binding by retaining only the amino acid sequences that associate with the Fc region of IgG. Each Protein G molecule can bind 3 IgG molecules.The recombinant Protein G is covalently linked to 4% cross-linked Agarose . Each ml of Protein G agarose is coupled with approximately 2mg of recombinant Protein G and can bind more than 20mg of human IgG. The main parameters of this product are listed below
Precautions： Do not freeze this product.This product must be fully resuspended by inverting the tube several times prior to use.This product contains a small amount of preservative and will not interfere with routine antibody purification and immunoprecipitation. However, if subsequent enzyme activity assays are involved, we recommend washing the product three times with an appropriate solution such as TBS before use to fully eliminate any possible interference from the preservative.Protein samples should always be placed at 4ºC 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. Antibody purificationa. Preparation:(a) Filter all solutions used with 0.45 µm or 0.2 µm 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 G Agarose.(d) Wash and equilibrate the purification 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.(e) We recommend diluting the protein sample at 1:1 or higher, or dialyzing the protein sample with PBS for better binding to this product.b. Antibody purification:(a) Load the 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 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 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 Gssays.c. Regeneration of purification columns:(a) Wash the purification column with at least 5 times the bed volume of PBS.(b) Wash in situ with at least 2 times the bed volume of 0.1 M or 0.5 M NaOH for at least 10 minutes.(c) Immediately wash the purification column with at least 5 times the bed volume of sterile and degassed PBS to neutral pH and store the regenerated purification column at at 4℃.Note: The cleaning and regeneration of purification columns should be performed immediately after elution. In general, we recommend cleaning once after 5 cycles of use, or washing once with 0.1M NaOH in situ after each use, once with 0.5M NaOH in situ after 10 cycles of use.2. Immunoprecipitationa. 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 buffer .(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 approximately1µg of IgG of the same species as the IgG used for immunoprecipitation and 20 µl of well resuspended Protein G Agarose. Shake slowly at 4℃ for 30 minutes to 2 hours.Note: For example, if mouse IgG is used for subsequent immunoprecipitation, normal mouse IgG can be added in this step. By incubating with normal IgG and Protein G Agarose, the non-specific binding and background 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 G Agarose and shake slowly for 1-3 hours at 4℃. 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 G resin 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 G resin with 20-40 µl of 1X SDS-PAGE loading buffer by vortex or pipetting. Pulse-spin to collect droplet.(f) Boil at 100ºC for 3-5 minutes and take some or all of the samples for SDS-PAGE analysis. The sample can be stored at -20ºC for later analysis.3. 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.
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