Immune (Co-)Precipitation (IP/CoIP)

 

This technique is based on the specific affinity between antibodies and proteins. It captures target proteins and enriches them from complex samples by binding antibodies that specifically interact with the protein or antigen, and can also be used to identify interacting proteins or other biomolecules.

 

Agarose Beads VS Magnetic Beads

 

Agarose beads have been used as solid-phase supports in immune precipitation experiments for many years. With the development of experimental techniques, magnetic beads have increasingly replaced agarose beads and become the preferred choice for immune precipitation purification methods. Many researchers who are new to this field face the problem of choosing between these two methods. What are the differences between magnetic bead and agarose bead methods? What are their respective advantages and disadvantages?

Figure 1 Structure of Agarose Beads

Figure 2 Magnetic Beads

 

Differences:

 

1. Antibody Binding Capacity: Agarose beads (diameter 50-150 μm) have a significantly larger surface area than magnetic beads (diameter 1-4 μm), so individual agarose beads can bind more antibodies than magnetic beads. However, the volume density of magnetic beads is higher than that of agarose beads;
2. Binding Ability: Agarose beads have a porous, sponge-like structure that provides better antibody binding capacity than magnetic beads;
3. Non-specific Binding: Agarose beads exhibit more non-specific binding than magnetic beads. Any non-target protein or any part of an antibody in the sample may bind to them. Therefore, sample pre-treatment is crucial in the agarose bead method;
4. Antibody Loss: Due to the porous structure of agarose beads, antibodies can easily become trapped within them. Additionally, antibodies may be lost during the centrifugation and washing processes;
5. Experiment Time: The magnetic bead method allows for short incubation times at room temperature (limited to Absin abs9649 and abs955 comparisons);
6. Special Equipment: The magnetic bead method requires a magnetic rack.

 

abs9649 (Magnetic Bead Method) and abs955 (Agarose Bead Method) Common Issues:

 

The following questions are specific to the two Absin kits abs9649 and abs955

 

I. Sample Processing

1. Do PMSF need to be added to the lysis buffer in both kits, and how should it be used?

 

PMSF should be prepared separately for both kits. We recommend abs9146. Based on the usage amount, add 10 μL of PMSF (100 mM, dissolve 174 mg of abs9146 in sufficient isopropanol and make up to 10 mL, then aliquot and store at -20 °C) to every 1 mL of lysis buffer to achieve a final concentration of 1 mM. Mix well and use immediately (PMSF should be added just before use). For the magnetic bead method, prepare the buffer according to the instructions, and dilute the buffer with ultrapure water.

 

2. Can enzymatic digestion be used to lyse cells if there is no abs9649 ultrasonic homogenizer available?

 

Both enzymatic digestion and ultrasonication are acceptable. Enzymes should be prepared at the normal laboratory concentrations.

 

3. How to estimate the protein amount?

 

A BCA protein assay kit (abs9232) can be used.

 

4. If the protein concentration in my cell lysate is higher than the total protein concentration required by the kit, what reagent should I use for dilution?

 

PBS can be used for dilution.

 

II. How to reduce non-specific binding of agarose beads?

 

Multiple washes and sample pre-treatment (by extending the incubation time of Protein A and Protein G) can reduce background. However, it should be noted that if this step is chosen, the number of samples that can be processed with the Protein A and Protein G in the kit will be halved.

 

III. What instrument can be used for overnight incubation at 4 °C during the binding process?

 

A mixer like abs72030 or abs72019 can be used.

 

IV. If the abs955 beads cannot be aspirated, what should be done?

 

Before aspirating, make sure to cut the pipette tip. If the beads cannot be aspirated, add an equal volume of 20% ethanol, mix well, and then aspirate.

 

V. If I want to run native gels without denaturing electrophoresis, how should I choose the sample buffer?

 

Use a WB loading buffer that does not contain SDS. However, whether the proteins can be eluted without denaturation is uncertain, and we cannot guarantee the subsequent experimental results.

 

VI. What are the differences between the two elution protocols in the magnetic bead method?

 

Both elution protocols include the capture antibody and target antigen. In the low pH elution protocol, the antibody remains intact, whereas in the denaturing elution protocol, the antibody is dissociated into heavy and light chains. Choose the elution protocol based on your subsequent experimental needs.

 

 

Catalog No.	Product Name	Specifications
abs955	Immune (Co-) Precipitation (IP/CoIP) Kit	50T
abs9649	Immune (Co-) Precipitation (IP/CoIP) Kit (Magnetic Bead Method)	50T

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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