| Product Description | Protein G Agarose is mainly used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and purification of antibodies as well. Protein G is an immunoglobulin binding protein expressed by Streptococcal bacteria type C or G; Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42kDa. Protein G and Protein A 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 gel 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 Protein A, Protein G and Protein A/G agarose gel products to common immunoglobulin subclasses of human, mouse and rat and the total binding capacity of different species.SpeciesIgProtein AProtein GA+GHumanIgG1++++++++++++IgG2++++++++++++IgG3-++++++++IgG4++++++++++++IgA++-++IgD++-++IgE++-++IgM++-++MouseIgG1+++++++++IgG2a++++++++++++IgG2b+++++++++IgG3++++++++IgM+/--+/-RatIgG1-++IgG2a-++++++++IgG2b-++++IgG2c+++++IgM+/--+/-Total IgProtein AProtein GA+GHuman++++++++++++Mouse+++++++++Rat+/-++++Rabbit+++++++++++Goat-++++Chicken-++Cow++++++++++Guinea Pig++++++++++Hamster+++++Horse++++++++++Pig+++++++++Sheep+/-++++ ++++, Strong Binding ++~+++, Medium Binding +, Weak Binding +/-, Weak or No Binding -, No BindingThe recombinant Protein G in this product binds specifically to the Fc-terminus of most mammalian IgGs and has a molecular weight of 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. Protein G in this product is covalently linked to 4% cross-linked Agarose, Fast Flow. Each ml of this product contains approximately 2 mg of recombinant Protein G and can bind over 20mg of human IgG. The average diameter of the agarose beads is 90μm, and the recommended linear flow rate for protein purification is 50-400 cm/h, with a maximum pressure tolerance index of 0.3MPa. This product is a 25% agarose gel suspension (i.e., 0.25 ml settled agarose per ml of this product). It is prepared in a suitable solution and can be used directly for antibody purification and immunoprecipitation. Each ml of this product is sufficient for 50 IP assays.
Precautions： Do not freeze this product. This product must be fully resuspended by inverting several times prior to use.The Agarose Gel contains a trace amount of preservative, which does not affect routine IP assays and protein purification. But for special experiments that might be interfered by the preservative, the gel should be washed 3 times with appropriate solutions such as TBS prior to use.During the IP assay or protein purification, keep protein samples at 4℃ or on ice all the time 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.Immunoprecipitationa.Preparation of protein samples(a)For adherent cells in 10 cm cell culture dishes, aspirate the culture medium, wash the cells once with PBS, and then add 500 µl to 2 ml of lysis buffer to lyse the cells, such as the Cell Lysis Buffer for Western and IP (, P0013) and RIPA Lysis Buffers (, P0013B, P0013C, P0013D or P0013E).(b)For tissue samples, grind or shear into fine pieces and lyse directly.(c)For suspension cells, collect the cells by centrifugation, wash the cells once with PBS, and then lyse the cells by referring to the lysis method for adherent cells.Note: For detailed lysis method, refer to the instructions of different lysis buffers. If the protein concentration obtained is too high, the sample lysate can be diluted appropriately with lysis buffer or PBS. Conversely, less amount of lysis buffer should be used for cell lysis if higher protein concentration is desired.b.Removal of non-specific binding (optional)(a)Take 0.2-1 ml of sample lysate with 0.2-1 mg of protein, add approximately 1 µg of IgG from the same species as the antibody 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 pre-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 subsequent immunoprecipitation.c.Immunoprecipitation (IP)(a)Add 0.2-2 µg of primary antibody into the sample lysate for immunoprecipitation and incubate overnight at 4℃ with shaking gently.(b)Add 20 µl of fully resuspended Protein G Agarose and shake slowly for 1-3 hours at 4℃. The amount of Agarose can be adjusted to 40 µl to facilitate washing subsequently.(c)Centrifuge at 2500 rpm (approximately 1000×g) for 5 minutes or instantaneously at high speed and carefully remove the supernatant without disturbing the gel.(d)Wash the gel 5 times with the same buffer used for cell lysis. Resuspend the gel with 0.5-1 ml of lysis buffer each time. Centrifuge at 2500 rpm (approximately 1000×g) for 5 minutes or instantaneously at high speed and carefully remove the supernatant without disturbing the gel after each wash.(e)Add 20-40 µl of 1X SDS-PAGE loading buffer and resuspend the gel by vortex. Boil at 100℃ for 3-5 minutes.(f)Centrifuge and take the supernatant for SDS-PAGE electrophoresis. Unused samples can be stored at -20℃ for future use.2.Co-immunoprecipitationRefer to the method for immunoprecipitation, but co-immunoprecipitation (co-IP) must be performed with fresh, unfrozen protein samples. For immunoprecipitation, fresh protein samples are also preferred although frozen protein samples can be used.3.Antibody purificationa.Preparation(a)Filter the antibody sample with a 0.45 µm or 0.2 µm pore size filter membrane.(b)Degass the sample using the ultrasonic method or other proper methods.(c)Load an appropriate amount of Protein G Agarose into an empty purification column of proper size. (d)Equilibrate the purification column with 10-20 times the bed volume of TBS at a flow rate of 1ml/min controlled by a constant flow pump. Alternative, the column can also be equilibrated with TBS entirely by gravity.b.Antibody purification(a)Load the antibody sample onto the purification column.(b)After all the antibody sample has passed through the column, wash the column with 10-20 times the bed volume of TBS to remove unbound and non-specifically bound proteins. Whether the wash is complete can be determined by measuring the absorbance of the flow through at 280 nm.(c)After washing, elute the antibody using 10 ml of elution buffer (50 mM glycine, pH 2.7). Some antibodies binding strongly to Protein G Agarose can be eluted with 50 mM glycine, pH 1.9. Collect the flow through in separate tubes and determine the tubes containing the purified antibodies by protein concentration assay or other protein detection methods.c.Regeneration of purification columns(a)Wash the purification column with 10-20 times the bed volume of TBS to neutralize the Protein G Agarose(b)Preserve the regenerated Protein G Agraose in TBS at 4℃.Related Products：Cat. No.Product NamePack SizeP2006 Protein A Agarose 2ml P2009 Protein G Agarose 2ml P2012 Protein A+G Agarose 2ml P2015-2ml Protein A Resin2ml P2015-10ml Protein A Resin10ml P2015-50ml Protein A Resin50ml P2015-200ml Protein A Resin200ml P2017-2ml Protein G Resin 2ml P2017-10ml Protein G Resin 10ml P2017-50ml Protein G Resin 50ml P2017-200ml Protein G Resin 200ml P2019-2ml Protein A+G Resin2ml P2019-10ml Protein A+G Resin10ml P2019-50ml Protein A+G Resin50ml P2019-200ml Protein A+G Resin200ml P2024 Protein A Agarose Prepacked Column, Fast Flow1 P2025 Protein A Agarose Prepacked Column, Fast Flow1 P2026 Protein G Agarose Prepacked Column, Fast Flow1 P2027 Protein G Agarose Prepacked Column, Fast Flow1 P2028 Protein A+G Agarose Prepacked Column, Fast Flow1 P2029 Protein A+G Agarose Prepacked Column, Fast Flow1 P2051-2ml Protein A Agarose 2ml P2051-10ml Protein A Agarose 10ml P2051-50ml Protein A Agarose 50ml P2053-2ml Protein G Agarose 2ml P2053-10ml Protein G Agarose 10ml P2053-50ml Protein G Agarose 50ml
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