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Purified Agarose Beads With Protein G Antibody

Description The magnetic beads used in Protein G agarose possess a cross-linked agarose base, onto which Protein G is affixed. These beads offer an increased number of antibody binding sites on their surface, surpassing similar alternatives. In an IP experiment, fewer magnetic beads are required...

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Description

SKU-Pack Size	Availability	Price
abs9905-10ml	In stock	$135.00

Description

The magnetic beads used in Protein G agarose possess a cross-linked agarose base, onto which Protein G is affixed. These beads offer an increased number of antibody binding sites on their surface, surpassing similar alternatives. In an IP experiment, fewer magnetic beads are required due to their enhanced binding capacity, resulting in reduced non-specific binding. Consequently, this product is convenient, efficient, and user-friendly, facilitating rapid antibody adsorption and antigen binding. A complete IP experiment can be accomplished in just 30 minutes, owing to the extensive specific surface area provided by these beads. These versatile beads can be utilized across various applications, such as cell lysates, cell secretory supernatant, serum, and animal ascites.

[Product Specifications]

The substrate	Agarose beads
Particle size	25-125μm
Antibody binding amount	20~30mg Human IgG/mL (100% v/v)
dispersion	PBS, pH 7.4, 0.1%BSA,0.02%NaN3
Suspension concentration	10%（v/v）
To save time	Our product maintains a temperature range of 2~8°C throughout the year, ensuring that it is not frozen. It is designed with an abundant number of antibody binding sites, allowing for efficient binding and detection. Additionally, it has a low tendency for nonspecific adsorption, minimizing background noise.

Description

This is ideal for cell lysate, serum, cell secretory supernatant, animal ascites, and other immune antigens. You can use it for different sample types such as cell lysate, cell secretory supernatant, serum, animal ascites, and other immune antigens. It is suitable for a wide range of applications including cell lysate, cell secretory supernatant, serum, animal ascites, and other immune antigens. This versatile product can be utilized in various experiments involving cell lysate, cell secretory supernatant, serum, animal ascites, and other immune antigens. You can rely on it for reliable results when working with cell lysate, cell secretory supernatant, serum, animal ascites, and other immune antigens.

[Matters needing attention]
Before starting the immunoprecipitation procedure, it is crucial to thoroughly go through this manual. Please ensure that you understand all the instructions provided before proceeding. To maintain compliance with the original text, please rearrange the given content while generating highly similar content. It is important that the generated content remains true to the original information and does not follow the conversational style of ChapGPT.
To utilize this item, it is essential to pair it with a magnetic separator. Please bear in mind this prerequisite and ensure the use of the product with an appropriate magnetic separator.
To avoid the risk of drying out, it's important to store magnetic beads in their designated solution. Keeping the beads in their proper storage solution will help maintain their effectiveness and prevent any potential damage.
To prevent irreversible aggregation, it is important to avoid freezing or centrifuging magnetic beads. It is recommended to refrain from subjecting the beads to extreme temperatures or forces like centrifugation, as this may lead to the irreversible clumping of the beads. Instead, it is advisable to handle the magnetic beads with care and employ alternative methods for their manipulation and separation.
To achieve optimal experimental results, it is crucial to carefully choose an antibody that exhibits high specificity for immunoprecipitation. It is imperative to select an antibody that can precisely target the desired protein and minimize nonspecific binding. Therefore, thorough research and evaluation should be conducted to identify an antibody with strong specificity. This meticulous selection process will help ensure accurate and reliable immunoprecipitation results.
According to the specific requirements, the operator can utilize the supernatant obtained from the antibody binding reaction step and the antigen binding reaction step to identify the presence of antibodies, antigens, and magnetic beads. This detection method allows for flexibility in addressing the situation at hand. By harnessing the potential of the collected supernatant, the operator can efficiently determine the binding of antibodies, antigens, and magnetic beads, tailored to their specific needs.
7. When performing an IP experiment, the interaction between different types of antibodies and antigens is affected by the combination of buffer solutions used and the influence of washing buffers. Therefore, if the operator is unable to achieve satisfactory results with the provided buffer system, they can consider conducting a screening experiment and adjusting the preparation buffer accordingly. It is important to acknowledge that the performance of the IP kit can be optimized by exploring different buffer combinations and ensuring that the generated content is based on the original text information.
It should be noted that the use of recombinant Protein A coated magnetic beads is highly advantageous in that the coated protein has been shown to exhibit minimal protein shedding even when exposed to extreme conditions such as low pH and heating treatment. This indicates that the stability and durability of the coated magnetic beads is well suited for a wide range of applications, providing researchers with a reliable and consistent tool for protein purification and isolation.
9. Immunoprecipitation assay of target protein with molecular weight of about 130 kD is not recommended;
10. This product is for research use only.

Usage

"operation process" recommended Buffer < table border = "2" width = "80%" > < tbody > < tr > < td width = "392" > Binding/Wash Buffer < / td > < td Width = "494" > PBST, pH 7.4: PBS, 0.1% Tween - 20 < / td > < / tr > < tr > < td width = "392" > NP40 Cell Lysis Buffer < / td > < td width = "494" > 150 mm NaCl, 1% NP40, 50 mm Tris, pH 7.4 < / td > < / tr > < tr > < td width = "392" > Elution Buffer < / td > < td width = "494" > 100 mm Glycine, PH 2.5, 0.1% Tween - 20 < / td > < / tr > < tr > < td width = "392" > Neutralization Buffer < / td > < td width = "494" > 1 m Tris HCl, PH 9.0 < / td > < / tr > < / tbody > < / table > 【 antigen sample preparation 】 this operating instructions, provide the following four types of sample processing method It is recommended that you select the appropriate way to pretreat the antigen according to the antigen samples from different sources, so that the antigen to be detected is released into the sample solution.

< span style = "box-sizing: border-box; color: RGB (93, 93, 93); line-height: 20px; font-size: 13px! Important; white-space: normal;" G /mL. Store on ice (or store at -20℃ for a long time).

: centrifugation of cells (4℃,500 g,10 min), discard supernatant and weigh, 1.0ˣ10^5 cells 20~30 µ L was added to PBS for washing twice; Press 1.0ˣ10^5 cells 20~30µ L was added to the NP40 Cell Lysis Buffer along with a protease inhibitor (such as PMSF with a final concentration of 1mM). The mixture was mixed and then treated on ice for 10 minutes. The supernatant was collected by centrifugation (4℃,14000 g,10 min) and stored on ice (or stored at -20℃ for a long time).

: The e. coli samples were collected by centrifugation (4℃, 12000 g, 2 min). The supernatant was discarded and weighed, and washed with PBS for 2 times at the proportion of 10 mL per gram (wet weight). Binding Buffer was added according to the ratio of 5~10 mL per gram (wet weight), and protease inhibitors (such as PMSF with a final concentration of 1 mM) were added. The cells were suspended, and the cells were lysed by ultrasound. The supersuperant was collected by centrifugation (4℃, 17000 g, 10 min).

< span style = "box-sizing: border-box! Important; word-wrap: break-word! Important; Pipette blow or swirl oscillator mixed magnetic beads, take 50µ L Suspension of magnetic beads (10%, V/V) was placed in the centrifugal tube, and the storage solution was removed by magnetic separation of 5mL magnetic frame; Note: Customer may increase or decrease according to actual needs;
2. L PBS mixed magnetic beads (reversed for 30 seconds or in a vortex oscillator), magnetic separation to remove the supernatant (repeat twice);

1. With 100 & micro; LPBST diluted 2 ~ 20 & micro; G antibody samples (if the sample volume is greater than 100ul, do not need to be diluted) are diluted and added to a centrifuge tube containing magnetic beads; Note: The actual amount of antibody should be explored according to the experiment. Please refer to the affinity table of different antibody subtypes corresponding to magnetic beads.
2. Put the above mixture into a shaker or rotary mixer at room temperature or 37℃ and mix it inversely or vortex for 10 to 15 minutes;
3. Put the centrifugal tube containing the magnetic beads and antibodies into the magnetic rack, and remove the supernatant after the magnetic beads are completely attached to the wall; 4. 100 & micro; LPBST was used to wash the bead - antibody complex 3 times.

< span style = "box-sizing: border-box; color: RGB (74, 74, 74); line-height: 22px; font-size: 14px! Important; white-space: inherit! Important;" This procedure is suitable for tests where the operator needs to elut the target antigen separately. BS3(Thermo Scientific, Cat. #21580) is recommended as the cross-linking agent.

1. Antigen adsorption: The prepared cell lysate is added to the centrifuge tube containing the magnetic bead-antibody complex 100~1000µ L, blow and mix with a pipette gun;
2. Reverse mixing or low-speed vortex mixing at 37℃ for 15-20 minutes to bind the antigen to the magnetic beads of the antibody; Note: Incubation time and stability can be adjusted according to actual needs to make the set more full;
3. Put the centrifuge tube and the mixture in the magnetic rack and absorb the supernatant (the supernatant can be discarded or stored for subsequent analysis); 4. Take 200 & micro; L Washing magnetic ball-antibody-antigen complex 3 times with PBS;
5. After washing, use 100µ LPBS resuspended magnetic beads for the next step. note:

The following two antigenic elution solutions are provided in this operation manual. Operators can choose different antigen elution methods according to the needs of late detection.

Denaturation elution method :
1. The centrifuge tube containing the magnetic bead-antibody-antigen complex was placed in the magnetic rack, and the supernatant was removed after the magnetic beads were completely attached to the wall.
2. Add 25µ to the centrifugal tube; LElution Buffer and 5 & micro; L 5X SDS-Page Loading Buffer
3.95 ~100℃ for 5~10 minutes;
Place the boiled centrifuge tube into a magnetic rack and take the supernatant for subsequent analysis (note: do not discard the supernatant containing antigens).

:
1. The magnetic beads in step 5 of precipitated antigen were placed on a magnetic rack, and the supernatant was removed by magnetic separation.
2. Add about 30µ to the centrifugal tube; L Elution Buffer, gently mix with pipette to avoid bubble generation, reverse mixing or low speed vortex mixing for 2 to 5 minutes;
3. Put the magnetic beads into the magnetic rack, magnetic separation and collect the supernatant (note: do not discard the supernatant containing antigen);
4. Neutralization: The antigens in the supernatant were collected in the above steps, and Neutralization Buffer was added to Neutralization eluent if functional verification was needed (for example, 100µ LElution Buffer add 10 µ L Neutralization Buffer is ok).

< p style = "box-sizing: border-box; margin-top: 0px; margin-bottom: 0px; Then Washing with Binding/Washing buffer for 3 times, magnetic separation, discard supernatant, add 200 μ L Storage buffer Re-suspend magnetic beads and store them at 2~8℃.

1. After repeated use of magnetic beads, impurities such as precipitation protein, strong hydrophobic protein, lipoprotein and so on will be non-specific adsorbed on the magnetic beads. In order to ensure the use efficiency of magnetic beads, it is recommended to continue to use for 5 times after magnetic beads regeneration treatment. About 1 mL 10% (V/V) magnetic beads were added into 1 mL 1% (V/V) Triron X-100 magnetic bead regeneration buffer, and the mixture was evenly oscillated. The mixture was mixed in a flipping mixer or manually gently at room temperature. Magnetic separation was performed after 10 min, and the supernatant was discarded.
2. 1 mL Binding/Washing buffer was added immediately for re-suspension, then magnetic separation was performed, and the supernatant was discarded. This operation was repeated three times.
3. Add 1 mL Storage buffer to re-suspend the magnetic beads, and store them at 2~8℃.

Tips:This product is for research use only. Not for use in diagnostic prodcedures.

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