Applied Biosystems™ Dynabeads™ MAX EPEC/VTEC O121 Kit

Catalog No.	Quantity
50-114-8242	200 Reactions

Catalog No. 50-114-8242 Supplier Applied Biosystems™ Supplier No. A14684

Description

Dynabeads MAX EPEC/VTEC O121 Kit

The Dynabeads™ MAX EPEC/VTEC O121 Kit allows for the rapid, selective concentration of E. coli O121 serogroup bacteria in food and environmental samples to improve downstream detection via PCR or culture methods.

Improve detection sensitivity in PCR sample analysis

Reduce background in selective agar detection

Perform the method manually or automate using the BeadRetriever™ System

Improve Sensitivity and Reduce Background
Highly specific antibodies attached to paramagnetic beads are incubated with an aliquot of pre-enriched sample. The beads bind and concentrate the target bacteria from non-target organisms. Washing steps help limit the carryover of inhibitors and matrix-associated particulates. These steps combine to improve the sensitivity of PCR-based detection while reducing background for plating methods.

Manual or Automated Processing
The immunomagnetic separation (IMS) steps can be performed manually with magnets and mixers or automated using the BeadRetriever™ System.

Sensitivity
Following the Dynabeads™ MAX EPEC/VTEC O121 protocol, the presence or absence of one viable E. coli O121 can be determined in the sample sizes described if this one E. coli cell is able to replicate and is not masked by resident background flora.

NOTE: Lot 1375851 of this product contains only 1 vial of paramagnetic beads (1 mL each) sufficient for 100 reactions. This batch follows a slightly modified protocol (see use instructions in document part number MAN0007304 Revision 1.0).

Specifications

• Content And Storage: Store in refrigerator (2–8°C). DO NOT FREEZE.
• Product Type: EPEC/VTEC Kit
• For Use With (Equipment): Dynabeads™ MPC™, KingFisher mL Food Protection Purification System
• Prep Scale: 150 μL output (automated prep), 100 μL output (manual prep)
• Product Line: Dynabeads
• Quantity: 200 Reactions

Frequently Asked Questions (FAQs)

What are Dynabeads magnetic beads?

Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads magnetic beads M-280 and 20% by weight in Dynabeads magnetic beads M-450. The Dynabeads magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.

Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.

The Dynabeads magnetic beads are available in three different sizes: 4.5 µm (M-450 beads), 2.8 µm (M-270/M-280 beads) and 1 µm (MyOne beads). as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

What are the benefits of the Dynabeads magnetic beads microbiology enrichment products?

Dynabeads magnetic beads microbiology enrichment products offer the following advantages:

Simple protocol
Cost effective
Shelf-stable reagents
Easy QC
High sensitivity
Saves time (for Salmonella in processed food, the assay is 24 hours faster compared to standard methods because immunomagnetic separation (IMS) replaces conventional selective enrichment; also saves time in confirmation procedure because you already have the colony isolated)

What do the designations M-280, M-270, and MyOne mean on Dynabeads magnetic beads?

The M stands for magnetic. M-280 refers to hydrophobic 2.8 micron beads, while M-270 refers to hydrophilic 2.8 micron beads. MyOne refers to 1 micron beads. as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

What is the detection limit when using Dynabeads magnetic beads for immunoprecipitation (IP)?

Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase)-based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)-based detection system. When a sensitive detection system is used, detection will most likely be in the nanogram range. In some cases, pictograms of target can be detected.

What is the elution volume when using Dynabeads magnetic beads for immunoprecipitation (IP)?

Within practical limits, the elution volume can be scaled up or down to suit your experiment. However, volumes less than 10 µL become more difficult to work with. In addition, the amount of target is important. If you have a lot of beads with a lot of bound target in a small elution volume, your elution may not be very efficient. Typically, 15-100 µL of beads may be eluted in 30 µL. For efficient recovery of the antigen and/or binding partners, the elution volume should at minimum equal the volume of the beads.

How can I quantify the amount of antibody bound to Dynabeads magnetic beads?

There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure the concentration of antibody in the coupling reaction before and after antibody attachment. Either fluorescence measurements or absorbance at 280 nm can be used. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods. as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

How long should I incubate my antibody with the lysates?

Incubation time will depend on the immunogenicity of the primary antibody and its binding affinity with the specific antigens. For a good primary antibody, 30-40 minutes incubation should work well. If you are working with a poor antibody or a very low-abundance protein, you could try to increase binding by incubating overnight. However, this also increases the chance of background protein binding.

When should I covalently bind the antibody to the Dynabeads surface?

If the target protein has the same molecular weight as the heavy or light chain antibody, we would recommend covalently binding the antibody to the bead surface. This can be done by either crosslinking the antibody to the Dynabeads Protein A or G magnetic beads, or secondary coated beads, or by using one of the surface-activated Dynabeads magnetic beads. as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

What are the general advantages of using Dynabeads magnetic beads for protein isolation?

Using Dynabeads magnetic beads for protein isolation provides several advantages:

-Rapid binding kinetics: since the number of beads per volume for Dynabeads is approximately 1,000 times higher than for the same volume of a Sepharose slurry, the probability for Dynabeads magnetic beads to hit the target is far greater.

-Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
-Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
-Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
-Low antibody consumption: this is because Dynabeads magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
-Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.

Are Dynabeads magnetic beads compatible with dithionite, DTT, and EDTA?

No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin. as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Are Dynabeads magnetic beads compatible with Urea?

Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.

Are Dynabeads magnetic beads compatible with centrifugation?

Dynabeads magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells. as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

What are the benefits of using magnetic beads in immunoprecipitation (IP) experiments?

Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 µm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding.

High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein.

However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).

For Testing of Food and Environmental Samples Only.