PE/Elab Fluor^® 594 Mouse IgG3, κ Isotype Control[A112-3] (E-AB-F09753P)

Q1:Why centrifuge before use?

During the transportation of antibodies, antibodies will stick to the tube wall or cap due to turbulence. So after receiving the antibodies, moderate centrifugation will collect the antibodies on the tube wall or cap to the bottom of the tube to avoid the loss of antibodies.

Q2:What is the difference between the test-package and the weight-package of flow antibody products?

The usage of test-package antibodies is well designed and verified there is no need for an extra dilution before use. The weight-package antibody has a higher concentration, and requires a titration process for a suitable usage amount.

Q3:What is the concentration of primary antibody?

Our antibodies are concentrated, and the final concentration of the antibodies is not only related to the concentration of the antibodies themselves, but also to the abundance of the target protein in the sample. Therefore, the recommended dilution ratio for the antibodies provided in our instructions is a range, and the final dilution ratio needs to be determined through pre-experiments.You can also consult our technical team to get a recommendation of a suitable dilution ratio for your experimental conditions.

Q4:What does Isotype Control do? How to choose a suitable isotype control antibody?

Isotype control antibodies are used as the basis for determining negative and positive cells. It is necessart as a gating helper especially for the indicators with low expression or continuous expression.
The Isotype control was purified from the serum of non-immunized animals, it should be the same species source, same immunoglobulin and subtype, same fluorescein label, same dose and concentration as the stained monoclonal antibody.

Q5:What does IHC mean on the datasheet? Is it a frozen tissue section? How is it different from IHC-F?

IHC is an immunohistochemical experiment, and the sections used generally include IHC-P (paraffin tissue section) and IHC-F (frozen tissue section).

Q6:What auxiliary reagents are needed for staining?

For samples with erythrocyte, ACK lysis buffer (E-CK-A105) is needed;
For cells that are rich in Fc receptors, such as macrophages, Fc receptors blocking is necessary before staining with flow Antibody to reduce the non-specific signal. At present, we can provide human and mouse blocking agent, E-AB-F1236A Purified Anti-Human CD16 Antibody[3G8]. E-AB-F0997A Purified Anti-Mouse CD16/32 Antibody[2.4G2].
Cell staining buffer (E-CK-A107) is required in the process of cell staining.
For the detection of intracellular indexes, a fixation & permeabilization kit is needed (E-CK-A109) ; and for the detection of intranuclear indexes, a specific staining kit (E-CK-A108) is required.
Dead cell dyes are also used for flow cytometry of tissue samples such as tumors.

Q7:What are the requirements for centrifuge usage when preparing samples?

The centrifugal force of the centrifuge does not exceed 300g, the centrifugal speed does not exceed 3, and the centrifugal speed does not exceed 1, so as to avoid cell damage.

Q8:What are host and reactivity respectively?

Host refers to the species from which antibodies originate. Reactivity refers to species that have been experimentally proven to bind specifically to our antibodies.

Q9:The molecular weight in the literature is different from that in the manual. Why are there several molecular weights for the same index?

Because the same protein in different types of cells may have different post-transcriptional splicing bodies and post-translational modifications, such as glycosylation, ubiquitination, etc., the molecular weight is not the only constant. In addition, the protein You can find on the official Uniprot website that the molecular weights of different subtypes of proteins are different. Therefore, there will be a certain difference between the molecular weight found in the literature and the molecular weight of the antibody in the instructions. (You can check some literature references to confirm the molecular weight of the target protein in the target sample).

Q10:Many thanks for your valuable information, As isotype control helps notify nonspecific binding, how is it helpful in setting gates?

In this article, we recommend using FMO Combined isotype control as a negative control to set gates. However, FMO Combined isotype control is more complicated, so many experimenters will choose a simpler method and only use isotype control as a negative control to set gates. Isotype controls are used to eliminate non-specific binding of antibodies causing background staining to help find true negative cell populations.