When you choose antibodies, "nine big pits", have you recruited?

Recently, the young lady of our technical department received a lot of questions from users and friends, saying, "I don't know which antibody is suitable for the protein being studied ."

There are also users who often ask "The instructions in the manual can be used for ICC/IF , then I can do fluorescent imaging of the tissue? "

In fact, there are many “pits” on the road to choose the right antibodies. As long as you successfully avoid them, you can choose the antibody that is most suitable for your experiment.

Today, let's share with you " How to choose the right antibody ."

1. Protein name and subtype

Our salesmen often get some "plagiarized" English names or abbreviations. When checking, it is not sure whether the recommended ones are the targets that the user wants to detect. For this problem, it is recommended that you refer to the SwissProt protein database. The "Database Link" section of each abcam specification has a link to the gene and protein database for different species. The SwissProt is the database link for the protein. There is a filter box on the left side of the page, you can click on the part you are interested in.

Click on the Names&Taxonomy section to see the target's gene name and protein name, as well as the alternate name, the so-called "small name". See if the name you provide can be matched, so that the antibody is suitable.

In addition, regarding the protein subtype problem, you can also find the answer here. Click on the Sequences section to see that the protein has several subtypes, the specific amino acid sequence and molecular weight of each subtype. If you just do the WB experiment, You can estimate the results of your experiment, which is the position of the strip that may be detected.

Can't help but say that the SwissProt database is too powerful. The subcellular location that we often ask users - Subcellular location , post-translational modification - PTM/Processing part, specific modification sites and modification groups, can be viewed one by one. .

2, the application range of antibodies

Common immunological experiments, in the specification of abcam , mainly include the following applications: WB , IHC-P , IHC-Fr , IHC-FrFl , IHC-Wmt , IHC-FoFr , ICC , ICC/IF , IP , ChIP , ChIP/Chip , ELISA , EM , Flow Cyt, etc. I believe that you will see these English words in a fog, just an immunohistochemistry - IHC, you can subdivide so many ways of operation, and in different brands The meaning of different English words is not the same. It is recommended that you ask for the definition and warranty coverage of different brands before purchasing .

For example, in the abcam product manual , IHC-P is an immunohistochemical paraffin section, and is internally tested with an enzyme-labeled secondary antibody, which is detected by DAB colorimetry; IHC-FR is an immunohistochemical frozen section. For internal verification, it is detected by fluorescence imaging with fluorescent secondary antibody; IHC-FoFr is frozen perfusion slice; IHC-FrFI is frozen slice; ICC/IF or ICC are specific fluorescence imaging at the cellular level. It is worth noting here ICC / IF in the IF, many people will mistakenly believe that both organizations also include fluorescence imaging cells, resulting in the wrong antibody, can not make the results.

3. Sample species that can be recognized by antibodies

Common test species, often described as follows: Reactivity : Human , Mouse , Rat , Sheep , Rabbit, Chicken, Hamster, Cat, Dog, Carp, African green monkey, Opossum, Chinese hamster, Common marmoset, Meriones unguiculatus .

Sometimes you will see the following description : predicted: Cow, Pig, Mammals .

Please be sure to note: in abcam of the Prospectus, Reactivity to the back row of the species is the warranty, and predicted the column to the species is based on homology to the proteins in different species of matching values, make Speculated. Speculated species have not been laboratory-tested and cannot be guaranteed for reference.

4. Animal sources of antibodies

The animal source of the antibody is the host species of the antibody, and the common ones are Rabbit , Mouse , Rat , Chicken , Goat , Sheep, etc. When you purchase the antibody, it is recommended to select and detect the host species different from the sample species, that is, you detect the mouse. , try to select a non-mouse-derived antibody, antibody can be selected from sources r abbit.

5. Preparation of antibody immunogens

The immunogen in the specification, the Immunogen part, is often described as follows, Synthetic peptide within Human X aa 200-250. The exact sequence is proprietary. or Recombinant full length protein corresponding to X ( where X refers to the name of the protein) It is recommended that you select the appropriate antibody for your experimental purposes. If you do not have special experimental requirements, the full-length immunogenic sequence is a good choice.

5. Monoclonal and polyclonal antibodies

Users often ask, is monoclonal or polyclonal antibody good? In fact, before answering this question, we must first explain the meaning of the two. In simple terms, monoclonal antibodies can only recognize specific epitopes of antigens, while polyclonal antibodies can recognize multiple epitopes of antigens, so The monoclonal antibody has high specificity and relatively low sensitivity, and the polyclonal sensitivity is high and the specificity is low. In different experimental operations, the selection can be made according to the following table.

7, the clone number of the antibody

The clone number is the number assigned to the antibody produced by the monoclonal hybridoma cells. Each hybridoma cell clone produces a single pure homologous antibody. The antibody with the same clone number represents the same immunogen sequence when the antibody was prepared.

8, antibody conjugate

Common conjugates include enzymes, fluorescein, biotin, etc. Common enzymes include horseradish peroxidase-HRP, alkaline phosphatase- AP, etc. Common fluoresceins are traditional fluorescein FITC , APC , Pe , Pe-cy3 , Pe-cy5, etc., the new fluorescein includes Alexa Fluor® series and DyLight® series of fluorescein at various wavelengths. Generally, when performing multiple immunostaining, the primary antibody that directly labels fluorescein is considered. It is recommended that you verify the experiment first. The fluorescence microscope or flow cytometer configuration of the chamber instrument is chosen to match the excitation wavelength of the instrument or the fluorescent antibody of the channel to minimize spectral overlap.

If you need to find common combinations in double-labeled and triple-labeled immunofluorescence staining experiments, we recommend the following:

9, antibody verification

Here we focus on the most convincing gene knockout validation, the KO validation, which uses the CRISPR/Cas9 KO human cell line of the haploid cell model . The knockout model is a true negative control and an excellent standard for antibody validation.

The gene knockout model understands the function of a particular gene by observing the phenotype of gene function loss, which is the power of the model. Currently, abcam has more than 1,600 knockout-verified antibodies, and more than 200 non-specific antibodies have been removed from the abcam catalog in the past year . Of course, knockout verification is only one of the many steps taken to improve antibody quality standards.

These are the problems that are often encountered on the road of antibody selection, and I hope to help you. Guangzhou Dongrui Technology Co., Ltd. and Guangzhou Xinjin Biotechnology Co., Ltd. are the first-level authorized agents of abcam in South China to protect your research and help you achieve your research mission faster.

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