Antibody & Protein FAQs

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.

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

First of all, customers need to estimate the dosage of antibody according to the antibody instructions & the results of pre-experiments. Different samples will have different antibody dilution ratios due to different antigen expression. For example, in the IHC test of an antibody, the pre-test confirmation is calculated according to the dilution ratio of 1: 50. Under normal circumstances, one slice needs to add 100μl of diluted antibody, that is, one slice needs 2μl of antibody, and 20μl can make about 10 slices.

The species mentioned in the antibody instructions can be confirmed, but there is no guarantee that the antibody can be used in unverified species, even if the sequence homology is high. Whether an antibody will recognize proteins in samples from other species involves many factors and we cannot predict. If there is no other choice, you must consider purchasing antibodies for use in unverified species. We recommend that you compare the immunogen sequence with your target protein sequence. The higher the homology, the greater the possibility of success. If the antibody you purchased is for use in unverified species or experimental applications, product replacement or refunds are generally not available.

General antibody instructions will list the types of experiments for which the antibody has been verified to be suitable (such as: WB, IHC, IP, IF, etc.). If the antibody instructions do not mention the application type, it does not mean that the antibody is not suitable. For this type of analysis application, it may be that we have not done the corresponding verification, or the verification sample is not very effective. Generally, if there is no application or the reactivity is not recommended for customers, please try to follow the verified ones listed in the instructions. Type to choose the right antibody for your experiment. If customers want to try it, they can consult technical support in advance or purchase small specifications for corresponding experiments. However, if the expected results are not achieved, product replacement or refunds are generally not available.

The secondary antibody is selected based on the source of the primary antibody and the customer's experimental needs. First look at the species origin and subtype of the primary antibody used in the experiment. Determine the secondary antibody based on the species source and subtype of the primary antibody: if the primary antibody is rabbit-derived IgG, then choose an anti-rabbit IgG secondary antibody, such as Goat Anti-Rabbit IgG; if the primary antibody is mouse-derived IgM, then choose an anti-mouse IgM secondary antibody, such as Goat Anti-Mouse IgM. Marker selection. Common markers include HRP, Biotin, fluorescein, etc. ELISA commonly uses Biotin-labeled secondary antibodies, followed by a streptavidin-labeled HRP for further signal amplification, and TMB substrate for color development; WB mainly selects HRP-labeled secondary antibodies and uses ECL substrate luminescent solution for color development; IHC generally chooses HRP-labeled secondary antibodies. It is recommended to use an immunohistochemistry kit (E-IR-R217 or the latest E-IR-R220/221), and generally use DAB substrate solution for color development; IF can choose secondary antibodies labeled with different fluoresceins according to experimental needs, such as FITC, Cy3, PE, etc. (try to avoid the autofluorescence of cells), and detect the fluorescence through a fluorescence microscope.

The use of positive control is the basis for determining whether the antibody and detection system work normally in an experiment, and it is also a basis for determining whether the protein to be detected exists in the sample to be detected! Especially when the detected sample is uncertain whether the protein to be detected is expressed. Therefore, when there is no positive result in the experiment, it is best to choose the appropriate positive control, and most of the instructions have recommended positive controls; Or you can search according to the SwissProt or Omnigene database in the instructions, these databases often list the tissues in which the protein is highly expressed, and these samples can be used as suitable positive controls.

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).

The repair conditions are generally high temperature and high pressure or microwave heating repair. For example: Antigen retrieval (high pressure method): Add 10 mmol/ml citrate buffer (pH 6.0) sufficient to submerge the slices in the pressure cooker (preparation: dissolve the antigen retrieval solution in the corresponding volume of ddH2O, mix well), and heat When it boils, place the slices on a heat-resistant plastic slicing rack, put them into the pot, cover the lid, fasten the pressure valve, continue heating, set the holding pressure for 4 minutes, open the vent valve to deflate after the time is up, and the pressure returns to zero. Open the lid, take out the inner pot and let it cool to room temperature. After the solution has cooled to room temperature, take out the slices (about 30 minutes). Antigen retrieval (microwave method): Add 10 mmol/ml citrate buffer (pH 6.0) sufficient to submerge the slices in a beaker (preparation: dissolve the antigen retrieval solution in the corresponding volume of ddH2O, mix well), and heat to boiling , place the slices on a heat-resistant plastic slice rack, put them into a beaker, heat over medium-low heat for 30 minutes, take them out, leave them to cool at room temperature, and take out the slices after the solution cools to room temperature (about 30 minutes). For the selection of IHC repair solution, it is recommended to refer to the antibody instruction manual, or refer to the following scheme: for human samples, it is preferred to use EDTA repair solution with pH 9.0 (clinical samples); for rat/mouse samples, it is preferred to use citric acid with pH 6.0 Repair fluid (scientific research sample).

Bovine serum albumin (BSA) is a monomeric protein is the main component in the blood plasma of cows (38g/1000ml), with a molecular weight of 68kD. Isoelectric point 4.8. Bovine serum albumin is widely used in biochemical experiments, such as as a blocking agent in western blot. If the customer wants to use it as a blocking protein for ELISA experiments depends on the customer's experimental system. Different BSAs have significant differences in ELISA experiment blocking, and the client can judge through pre experiments.

Dilute with TBST, BSA concentration 2-5%.