Immunoprecipitation Kit (Protein A Agarose), 50T

Product Information

Product Description/Introduction

IP or Co-IP is a common experimental technique for studying protein or protein-protein interactions (PPIs) by using specific antibodies and a medium that binds antibodies (e.g. Protein A/G Agarose or Protein A/G Magrose), or directly using a medium coupled with specific antibodies (e.g. agarose or magnetic beads), and then isolating the antigen-antibody complexes from the complicated protein samples by centrifugation or magnetic force, so as to achieve the purpose of isolation and purification of the target proteins, which can subsequently be used for Western blot or mass spectrometry. Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42 kDa. Protein G is an immunoglobulin binding protein expressed by Streptococcal bacteria type C or G. Protein A and Protein G are functionally similar and can bind specifically to mammalian immunoglobulin (Ig). Recombinant Protein A and G with appropriate modifications bound to agarose can be used for immunoprecipitation or antibody purification. Protein A agarose are suitable for the immunoprecipitation of human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, while Protein G agarose beads are suitable for the immunoprecipitation of human IgG1, IgG2, IgG3, IgG4, mouse IgG1, IgG2a, IgG2b, IgG3, rat IgG1, IgG2a, IgG2b, IgG2c polyclonal antibodies. (See Table I for specific information)

This product adopts the self-developed and produced Protein A labelled agarose, compared with similar products in the market, this product binds antibodies more efficiently and has a lower non-specific binding rate, together with the optimized buffer, it can be convenient and efficient for immunoprecipitation experiments; it can be widely used in the isolation and purification of the target proteins in samples such as cell lysates, cellular secretion supernatants, serum, ascites, etc.; This kit provides two elution methods (denaturing elution and acid elution) to elute the target protein, especially the acid elution will not contain the light and heavy chains of the antibody, which can effectively solve the problem of the interference of the antibody's heavy and light chains in the immunoprecipitation and protein immunoblotting experiments.

Product Information

Storage and Shipping Conditions

Ship with wet ice; 5×SDS-PAGE loading buffer (reduced) should be stored at -20 and other at 2-8 for 12 months.

Product Components

Additional Reagents Required

Assay Protocol/Procedures

1. Preparation of the kit

a) Refer to the table below, prepare the relevant reagents at a ratio of 100-500 μL of lysate per sample;

b) Preparation of IP lysate containing protease inhibitor: Refer to the above table, use 100-200 μL of IP lysate containing protease inhibitor per 0.5-1 million cells for lysis; mix the IP lysate with 50x Cocktail protease inhibitor (G2006-250UL is recommended) at a ratio of 50:1, for example, add 20 μL of 50x Cocktail protease inhibitor into 1 mL of IP lysate, then 1 mL of IP lysate containing protease inhibitor will be obtained; the prepared IP lysate containing the inhibitor should be placed in an ice bath or at 4

c) Note: If the target protein of the immunoprecipitation involves phosphorylation or acetylation modification, phosphatase inhibitor or deacetylase inhibitor should be added (self-provided); IP lysate containing inhibitor should be prepared prior to use, and should not be frozen and retained for subsequent use.

d) Preparation of 1×TBS: Dilute 10×TBS buffer with ultra-pure water to 1×TBS. For example, Add 1 mL of 10×TBS buffer to 9 mL of ultra-pure water, which is 1×TBS buffer after mix well.

e) Preparation of 1x SDS-PAGE loading buffer (reduced): an appropriate amount of 5x SDS-PAGE loading buffer (reduced) was diluted 5 times with ultra-pure water to make 1x SDS-PAGE loading buffer (reduced); for example, mix 0.2mL of 5x protein buffer (reduced) with 0.8 mL ultra-pure water, which is 1x SDS-PAGE loading buffer (reduced).

2. Antigen sample preparation

Immunoprecipitation or immunoprecipitation experiments should be carried out immediately after samples lysed, if not, the samples can be stored in the refrigerator at -20 or -80, but freezing and thawing may affect protein-protein interactions; all sample lysis steps should be operated in an ice bath or at 4 in order to minimise the degradation of protein , and a certain amount of the sample should be taken as an Input or a Total after the sample has been prepared for subsequent detection by Western Blot.

For tissue samples:

a) The tissues should be rinsed in pre-cold PBS (G4202 recommended) to remove excess blood thoroughly. Weigh and mince into small pieces in homogenizer on ice.

b) Add the IP lysate containing protease inhibitor at a ratio of 100-200 μL per 10-20 mg of tissue, or reduce the amount of IP lysate if higher concentrations of protein are required.

c) Homogenise with a glass homogeniser or handheld homogeniser, or use our self-developed KZ-III-F low-temperature grinder for full grinding.

d) The homogenate was transferred to a 1.5 mL centrifuge tube, shaken and mixed, and ice-bathed for 30 min, repeat blown with a pipette every 10 min to ensure complete lysis of the tissue cells.

e) Centrifuge at 12,000 x g for 5 minutes at 4, discard the precipitate and aspirate supernatant for subsequent immunoprecipitation or immunoprecipitation experiments.

For adherent cell samples:

a) If necessary, the cells can be washed 2-3 times with PBS, absorb the residual liquid thoroughly at the last time.

b) Scrape the cells with a cell scraper or trypsin digest the cells to make them fully suspended, collect them into a 1.5 mL centrifuge tube and centrifuge at 1,000 x g for 5 min at 4, discard the supernatant to collect the cell precipitate.

c) Add 100-200 μL of IP lysate containing protease inhibitor per 0.5-1 million cells (equivalent to one well of a 6-well plate); blow appropriately and ice bath for 3-5 min to fully lysed the cells, and there should be no obvious cellular precipitation after fully lysed; if the amount of cells is larger, it is recommended that the cells be divided into 0.5-1 million cells/tube to fully lysis.

d) After sufficient lysis, centrifuge at 12,000 x g for 5 minutes at 4, discard the precipitate and aspirate supernatant for subsequent immunoprecipitation or immunoprecipitation experiments.

For suspend cell samples:

a) Centrifuge at 1,000 x g for 5 min at 4°C to collect the precipitate; if necessary, wash once with PBS, then aspirate the residual liquid and gently vortex to disperse the cells as much as possible.

b) Add 100-200 μL of IP lysate containing protease inhibitor per 0.5-1 million cells (equivalent to one well of a 6-well plate); blow appropriately and ice bath for 3-5 min to fully lysed the cells; if the amount of cells is larger, it is recommended that the cells be divided into 0.5-1 million cells/tube to fully lysis.

c) Ice bath for 5 min, centrifugation at 12000 g 4 for 5 min, take the supernatant, that is, the total protein solution, which can be used for subsequent immunoprecipitation or immunoprecipitation experiments and so on.

For bacterial or yeast samples:

a) Take 1 mL of bacterial or yeast solution and centrifuge to remove the supernatant. If necessary, the cells can be washed once with PBS, absorb the residual liquid thoroughly at the last time. Gently vortex or flick the bottom of the tube to disperse the cells as much as possible.

b) Add 100-200 μL of IP lysate containing protease inhibitor and blow gently to fully disperse the bacteria or fungi.

c) Ice bath for 10 min, for better lysis, bacteria and yeast can be digested with lysozyme and wall-breaking enzyme (self-provided) respectively, and then lysed with IP lysate containing protease inhibitors.

d) Centrifuge at 12,000 x g for 5 minutes at 4, discard the precipitate and aspirate supernatant for subsequent immunoprecipitation or immunoprecipitation experiments.

3. Agarose preparation

a) Gently resuspend the SweAgarose Protein A beads to form a homogeneous bead dispersion as much as possible, add 20 μl of well-mixed bead dispersion for every 500 μl of sample, take an appropriate amount of agarose beads into a clean EP tube and add 1x TBS to a final volume of 0.5 mL (20 μl of bead dispersion for each sample is used in the following immunoprecipitation steps as an example).

b) Gently resuspend the beads, centrifuge at 6,000×g for 30s at 4, carefully remove the supernatant and repeat the above steps twice.

c) The beads were resuspended with 1x TBS according to the volume of the initial bead dispersion.

4. Antibody binding to SweAgarose Protein A beads

a) Antibody preparation: dilute the antibody with 1x TBS to prepare the antibody working solution according to the dilution ratio recommended in the antibody instructions, or prepare the antibody into an antibody working solution with a final concentration of 5-50 μg/mL, which can be prepared on ice; optional: use normal IgG of the same antibody species to prepare normal IgG working solution with the same dilution ratio or final concentration of 5-50 μg/mL, remove non-specific binding or serve as a negative control. The normal IgG of the same species means that if the antibody used in subsequent immunoprecipitation is mouse IgG, an appropriate amount of normal mouse IgG can be diluted with 1x TBS in this step to reduce the background or as a negative control.

b) Antibody adsorption: Separate the SweAgarose Protein A beads prepared in step 3 by centrifuge at 6,000 x g for 30s at 4, aspirate the supernatant, add 500 μL of antibody working solution or normal IgG working solution, resuspend and then incubate for 15-60 min at room temperature on a turnover mixer.

Note: It is also possible to incubate the SweAgarose Protein A beads prepared in step 3 directly with an appropriate amount of antibody or normal IgG.

c) Beads separation and washing: Separate the incubated beads by centrifuge at 6,000 x g for 30s at 4, aspirate the supernatant, add 500 μL of 1×TBS, resuspend the SweAgarose Protein A beads by gently blowing with a pipette, centrifuge at 6,000 x g for 30s at 4, remove the supernatant, repeat the washing for three times, and resuspend the beads with 1×TBS in the same amount as the initial volume.

Note: If the beads are agglomerated or flaky during incubation and washing, it is a normal phenomenon and will not affect the experimental results.

5. Immunoprecipitation (IP):

a) Removal of non-specific binding (optional): the SweAgarose Protein A beads prepared in step 4, which bind normal IgG, are incubated with the samples at 4°C for 60 min and separated by centrifuge at 6,000 x g for 30s at 4, and the supernatants are used for the subsequent experiments; the purpose of this step is to remove the proteins that bind non-specifically to normal IgG.

b) Incubation of samples with SweAgarose Protein A beads conjugated with antibody or normal IgG: add SweAgarose Protein A beads conjugated with antibody or normal IgG at the ratio of 20 μl of bead suspension for every 500 μl of protein sample, place on a side-oscillating shaker or a rotary mixer, and incubate for 2 hours at room temperature or overnight at 4ºC.

Note 1: If the beads are agglomerated or flaky during incubation and washing, it is a normal phenomenon and will not affect the experimental results.

Note 2: Alternatively, the appropriate amount of antibody or normal IgG can be incubated with the sample for 1-2 hours at room temperature or overnight at 4°C, and then 10-20 μL of agarose bead suspension can be added and incubated for 60 min at room temperature.

c) Centrifuge: after incubation, centrifuge at 6,000 x g for 30s at 4 to remove the supernatant.

Note: Retain a portion of the supernatant to test the effect of immunoprecipitation.

d) Wash: Add 500 μl of 1×TBS, gently blow the resuspended beads with a pipette, centrifuge at 6,000 x g for 30s at 4 to remove the supernatant and repeat the wash three times.

Note: You can also assay the OD280 of the washing buffer to determine whether the washing is complete, if the OD280 is more than 0.05, the number of washing times should be increased appropriately.

6. Elution

Depending on the characteristics of the target protein and the requirements of subsequent experiments, one of the following methods can be selected for elution.

a) Denaturing elution method: Samples eluted by this method are suitable for SDS-PAGE. Add 25 µL of 1× Protein Sampling Buffer (Reduced) to the tube after centrifuge, heat at 95°C for 5 min, and then centeifuge to collect the supernatant for Western blot detection.

b) Acid Elution: The sample eluted by this method retains its original biological activity and can be used for post-functional analysis. Add 20 µL of Acid Elution Buffer to the tube after centrifuge, mix well and incubate at room temperature for 10 min, then centrifuge to collect the supernatant into a new EP tube, and immediately add 2 µL of Neulization Elution Buffer to adjust the pH of the eluted product to neutral, which can be used for post-functional analysis.

Note: The user can adjust the amount of elution buffer added according to the desired volume.

Note

1. Do not store SweAgarose Protein A in the kit at -20 or freeze and thaw repeatedly, otherwise it will affect the performance of magnetic beads and cause unnecessary experimental errors.

2. Please read these instructions carefully before performing the immunoprecipitation procedure.

3. If the immunoprecipitated target protein involves phosphorylation or acetylation modification, appropriate phosphatase inhibitors and deacetylase inhibitors should be provided.

4. Maintain beads at pH 6-8, avoid high speed centrifugation, drying or freezing, which may cause agglomeration of the beads.

5. The beads should be mixed thoroughly before use, the mixing operation should be gentle and should not be subjected to violent swirling and shaking to avoid denaturation of the antibody.

6. Positive and negative controls (SweAgarose Mouse IgG) are recommended in immunoprecipitation.

7. Protein samples should be purified as soon as possible after collection and always placed at 4°C or in an ice bath to slow down protein degradation or denaturation.

8. Agarose beads may gather when use with acid elution, which is a normal phenomenon and does not affect the normal use of magnetic beads.

9. This product is restricted to scientific research use by professionals, and is not to be used for clinical diagnosis or treatment, food or medicine, or stored in an ordinary residence.

10. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves.

11. The binding capacity of Protein A and Protein G to antibodies of different generic sources and subtypes is shown in Table I.

Table I

For research use only!

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