| Product Description | Aladdin's Rabbit IgG Agarose, also known as Rabbit Normal IgG Agarose, Rabbit IgG IP Agarose, or Rabbit IgG Affinity Gel, is covalently linked with the high-quality unlabeled non-specific rabbit IgG. This product is commonly used as a negative control for rabbit-derived antibody agarose gels such as Anti-Flag Affinity Gel in immunoprecipitation (IP), co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and other immunoprecipitation-related assays.This product can also be used to reduce background by excluding non-specific binding of IgG itself to some proteins or other biological molecules.This product has high IgG binding capacity, with low non-specific adsorption. Per ml of settled gel contains approximately 5mg of IgG, which is ideal for removing non-specific binding. This product basically does not recognize any antigens. Main parameters of the Rabbit IgG Agarose
Precautions： This product must be fully resuspended by inverting the tube several times prior to use.This product contains a small 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.For immunoprecipitation assays, it is recommended to include both positive and negative controls. This product can be used for negative controls.Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can also be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use , Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X) , Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , etc.The non-specific binding capability of this product can be affected by high concentrations of DTT, mercaptoethanol, and guanidine hydrochloride, but it is applicable in Cell lysis buffer for Western and IP , RIPA Lysis Buffer , and NP-40 Lysis Buffer . For the main features and differences of various lysis buffers produced by and the selection of lysis buffers, please refer to the website at http://www.aladdin-e.com/support/lysis-buffer.htm.If the insoluble substance in protein samples can not be removed completely by centrifugation, samples can be filtered with a 0.45μm filter before performing the assay.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.Preparation of protein samplesa.Lysis cells or tissues with appropriate lysis buffer, such as ’s Cell Lysis Buffer for Western and IP . Under certain circumstances, ’s RIPA Strong Lysis Buffer , RIPA Medium Lysis Buffer , or RIPA Weak Lysis Buffer can be used. Other lysis buffers with a pH6-8 can also be used.a.After lysis and centrifuge, keep the supernatant at 4℃ or on ice for subsequent use. We recommend performing the subsequent procedures on the same day. Otherwise, make aliquots and store them at -80℃ for future use.2.Preparation of Rabbit IgG AgaroseAs the Rabbit IgG Agarose is stored in a protective solution containing 50% glycerol and a small amount of preservative, it needs to be washed properly before adding protein samples.a.Gently resuspend the Rabbit IgG Agarose, Transfer 10-20μl of gel suspension per 500μl of sample into a new centrifuge tube .Note: It is easier to aspirate the gel suspension using a wide orifice pipette tip (e.g., by cutting off part of the tip with scissors).b.Add 1X TBS (, ST661/ST665) to a final volume of approximately 0.5ml, and gently resuspend the Rabbit IgG Agarose by pipetting or vortex. Centrifuge at 6000×g for 30 seconds at 4℃, carefully remove and discard the supernatant. Repeat this step twice.c.Resuspend the Rabbit IgG Agarose with 1X TBS equal to the initial volume of gel suspension (e.g., if 10μl of gel suspension is taken, add 10μl of 1X TBS).3.Protein bindingd.Add 20μl of washed Rabbit IgG Agarose per 100μl of protein sample, mix well, and incubate at 4℃ for 1-2 hours or overnight at 4℃ with gentle shaking on a rotary mixer.e.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and carefully remove the supernatant.Note: A portion of supernatant can be reserved in a clean microfuge tube for examination of the binding results.f.Gently resuspend the Rabbit IgG Agarose in 500μl of 1X TBS and place in an ice bath on a shaker for 5 minutes. Centrifuge at 6000×g for 30 seconds at 4℃ and carefully remove the supernatant. Repeat the wash thrice, then keep the sample on ice for elution. 4.ElutionBased on the features of the target protein or further usage, one of the following three elution methods can be used. The following protocols are provided for eluting target proteins from the Anti-Flag Affinity Gel (, P2271). The Rabbit IgG Agarose negative control performed in parallel should be eluted in the same manner. 1)Elution with competitive peptide: This is a non-denaturing elution method with a high elution efficiency, and the eluted proteins do not contain the light and heavy chains of Flag antibody. a.Preparation of 3X Flag elution buffer: Dissolve the 3X Flag Peptide (, P9801-25mg) in 1X TBS buffer to a final concentration of 150μg/ml, or dilute the 3X Flag Stock Solution at 5mg/ml (, P9801) with 1X TBS buffer to a final concentration of 150μg/ml.b.For every 20μl of initial gel volume, add 100μl of 3X Flag Peptide elution buffer (150μg/ml). Resuspend the agarose gently and incubate for 30-60 minutes at room temperature on a rotary mixer , or 1-2 hours at 4℃. To improve the elution efficiency, increase the incubation time or repeat the elution. c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. The supernatant contains the Flag-tagged protein and its protein complex.d.Store the supernatant at 4℃ for immediate use, or -20℃/ -80℃ for long-term storage.2)Acidic elution. This method is non-denaturing, relatively fast and efficient. The eluted proteins also retain their original biological activity, which facilitates subsequent analysis and detection.a.Preparation of acidic elution buffer (0.1M Glycine-HCl, pH3.0) and neutralization buffer (0.5M Tris-HCl, pH7.4, 1.5M NaCl). b.For every 20μl of initial gel volume, add 100μl of acidic elution buffer. Resuspend the agarose gently and incubate for 5 minutes at room temperature on a rotary mixer.Note: The incubation time should not exceed 15 minutes.c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. Immediately add 10μl of neutralization buffer and mix well. d.To achieve the maximum elution efficiency, repeat steps b-c and combine the supernatant containing the Flag-tagged protein and its protein complex .e.Store the supernatant at 4℃ for or immediate use, or -20℃/-80℃ for long-term storage. Note 1: The elution efficiency of the acidic elution method might be lower than the other two elution methods.Note 2: The elution efficiency of the acidic elution method depends on characteristics of the target protein. To obtain a higher elution efficiency, the pH of the acidic elution buffer can be optimized from 2.5 to 3.1. The pH or amount of neutralization buffer needed to neutralize the eluates should also be adjusted appropriately.3)Elution with the 1X SDS-PAGE loading buffer. This is a denaturing elution method and the obtained protein samples are suitable for SDS-PAGE electrophoresis and Western blot analysis. a.Preparation of SDS-PAGE loading buffer: We recommend using ’s SDS-PAGE Sample Loading Buffer (2X) . Usually, the SDS-PAGE protein loading buffer contains a reducing agent such as DTT, and the eluted protein sample will contain the light chain and heavy chain of the Flag antibody.b.For every 20μl of initial gel volume, add 20μl of 2X SDS-PAGE sample loading buffer, mix well and heat at 95℃ for 5 minutes. c.Centrifuge at 6000×g or 4℃ for 30 seconds. Take the supernatant for SDS-PAGE electrophoresis or Western blot analysis.Note: The Agarose cannot be reused because SDS in the loading buffer destroys Flag antibodies.FAQ: Problem Possible Causes Solution Large amount of tagged protein found in the flow through. Binding time is not enough. If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. Column is overloaded. Reduce the amount of the sample added to the gel or increase the amount of gel. Tag is not accessible to gel. Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse thetag to the other terminus of the target protein. Gel has not been regenerated since last purification. Perform gel regeneration procedure prior to binding. Reagent compatibility problem. Dialyze the sample against TBS before purification procedure. The target protein has been degraded. 1. Prepare fresh lysates. Avoid using frozen lysates. 2. Perform purification at lower temperature, such as 4℃. 3. Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. Very few or notagged protein exists in the eluate. Protein is not completely eluted. Change elution methods. No target protein expressed. Make sure the protein of interest contains the tag by Western blot or dot blot analyses. Very low protein expression level. 1. Use larger volume of cell lysate. 2. Optimize expression conditions to raise the protein expression level. Washes are too stringent. 1. Reduce the number of washes. 2. Avoid adding high concentrations of NaCl to the mixture. 3. Use solutions that contain less or no detergent Incubation times are inadequate. Increase the incubation times with the affinity gel (from several hours to overnight). Interfering substance is present in sample. 1. Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided. 2. Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. Detection system is inadequate. If Western blot detection is used: 1. Check primary and secondary antibodies using proper controls to confirm binding and reactivity. 2. Verify that the transfer was adequate by staining the membrane with Ponceau S. 3. Use fresh detection substrate or try a different detection system. Multiple protein bands found in the eluate. The protein is not stable at room temperature. Purify the target protein at lower temperature, such as 4℃. Protein degradation due to proteases activity during purification process. Add protease inhibitors to cell lysate. Non-specific binding. 1. Prepare cell lysate again. 2. Add additional wash steps. Background is too high. Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. 1. Pre-clear lysate with Mouse IgG Agarose orRabbit IgG Agarose to remove nonspecific binding proteins. 2. After suspending beads for the final wash, transfer entire sample to a clean microcentrifuge tube before centrifugation. Washes are insufficient. 1. Increase the number of washes. 2. Prolong duration of the washes, incubating each wash for at least 15 minutes. 3. Increase the salt and/or detergent concentrations in the wash solutions. 4. Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins from the lysate during the initial centrifugation of the affinity gel complexes. Related Products：Cat. No.Product NamePack Size P2265-1ml Mouse IgG Agarose 1ml P2265-5ml Mouse IgG Agarose 5ml P2267-1ml Rabbit IgG Agarose 1ml P2267-5ml Rabbit IgG Agarose 5ml P2271-0.5ml Anti-Flag Affinity Gel 0.5ml P2271-2ml Anti-Flag Affinity Gel 2ml P2271-10ml Anti-Flag Affinity Gel 10ml P2282-0.5ml Anti-Flag Affinity Gel 0.5ml P2282-2ml Anti-Flag Affinity Gel 2ml P2282-10ml Anti-Flag Affinity Gel 10ml P2285-0.5ml Anti-Myc Affinity Gel 0.5ml P2285-2ml Anti-Myc Affinity Gel 2ml P2285-10ml Anti-Myc Affinity Gel 10ml P2287-0.5ml Anti-HA Affinity Gel 0.5ml P2287-2ml Anti-HA Affinity Gel 2ml P2287-10ml Anti-HA Affinity Gel 10ml P2289-0.5ml Anti-V5 Affinity Gel 0.5ml P2289-2ml Anti-V5 Affinity Gel 2ml P2289-10ml Anti-V5 Affinity Gel 10ml
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