There are a few things that need to be considered when choosing primary and secondary antibodies.

Secondary antibodies should not be specific for the animal you are testing in. For example, since you are using human cultured cells, almost any secondary will be fine unless it was generated in human cells, so mouse, rabbit, goat, chicken, rat etc should all work. Keep in mind, the secondary you choose depends on the host animal your primary antibody was generated from.

Primary antibodies are a little more complicated. The following is from the Abcam website.

The nature of the sample will dictate which antibody will work best. At least two aspects need to be considered:

1. The region of the protein one wishes to detect. Antibodies are generated by immunization of host animals with a variety of immunogenic substances including full-length proteins, protein fragments, peptides, whole organisms (for example bacteria), or cells. The immunogen is generally described on the datasheet (though in many cases an exact description of the immunogen is not available for proprietary reasons). If trying to detect a protein fragment or a specific isoform or region of the full-length protein, one needs to be sure to choose an antibody that is raised against an immunogen that is identical to or contained within the fragment or region. If trying to detect a cell surface protein on live cells by FACS, one needs to choose an antibody that is raised against an extracellular domain of the protein.

2. Processing of the sample. Some antibodies require samples to be processed or treated in a specific manner. For instance, many antibodies will only recognize proteins that have been reduced and denatured, presumably because this reveals epitopes that would otherwise be obscured by secondary and tertiary folding of the proteins. On the other hand, some antibodies will only recognize epitopes on proteins in their native, folded state.

Another thing to consider with primary antibodies is whether you want a monoclonal or polyclonal antibody. Polyclonal antibodies tend to be cheaper because they are raised against multiple epitopes on your protein and are therefore less specific. Monoclonal are more expensive and take longer to produce, however they are specific to a single epitope. Monoclonal antibodies are most useful if you are trying to identify specific isoforms or region of a full-length protein as stated in #1 of the Abcam section of this response.

I hope this answers your question and if you need more specific help, feel free to ask.

There are a few things that need to be considered when choosing primary and secondary antibodies.

Secondary antibodies should not be specific for the animal you are testing in. For example, since you are using human cultured cells, almost any secondary will be fine unless it was generated in human cells, so mouse, rabbit, goat, chicken, rat etc should all work. Keep in mind, the secondary you choose depends on the host animal your primary antibody was generated from.

Primary antibodies are a little more complicated. The following is from the Abcam website.

The nature of the sample will dictate which antibody will work best. At least two aspects need to be considered:

1. The region of the protein one wishes to detect. Antibodies are generated by immunization of host animals with a variety of immunogenic substances including full-length proteins, protein fragments, peptides, whole organisms (for example bacteria), or cells. The immunogen is generally described on the datasheet (though in many cases an exact description of the immunogen is not available for proprietary reasons). If trying to detect a protein fragment or a specific isoform or region of the full-length protein, one needs to be sure to choose an antibody that is raised against an immunogen that is identical to or contained within the fragment or region. If trying to detect a cell surface protein on live cells by FACS, one needs to choose an antibody that is raised against an extracellular domain of the protein.

2. Processing of the sample. Some antibodies require samples to be processed or treated in a specific manner. For instance, many antibodies will only recognize proteins that have been reduced and denatured, presumably because this reveals epitopes that would otherwise be obscured by secondary and tertiary folding of the proteins. On the other hand, some antibodies will only recognize epitopes on proteins in their native, folded state.

Another thing to consider with primary antibodies is whether you want a monoclonal or polyclonal antibody. Polyclonal antibodies tend to be cheaper because they are raised against multiple epitopes on your protein and are therefore less specific. Monoclonal are more expensive and take longer to produce, however they are specific to a single epitope. Monoclonal antibodies are most useful if you are trying to identify specific isoforms or region of a full-length protein as stated in #1 of the Abcam section of this response.

I hope this answers your question and if you need more specific help, feel free to ask.

In spite of being a classic technique, the diversity of products on the market left me confused.Thank´s all of you!

Some companies are known for great antibodies. I have had a lot of success of many antibodies from Cell Signaling Technology, as I do routine detection of phosphorylated and total proteins. Additionally, EMD Millipore and Abcam also make pretty good antibodies. Santa Cruz is very hit or miss for their antibodies: either they work great or they are super dirty. My recommendation is to look up papers and see what antibodies they are using and if they look good in their WB. What works well in one cell line will not always translate exactly to your cell type. (I work in primary human skin cells). Outside of my own experience, I think Kristyn and Ditte have defined very well what other parameters you should think about before ordering any antibody.

The protein I am working on is not published much but there are antibodies available for it by different companies. Surprisingly, antibodies manufactured by different groups have different size of the band for this protein. I have tried one manufacturer so far (Abcam) which turned out to be crap. Can any one suggest how I should select an antibody in this case? when there are so many discrepancies among manufacturers in size of the protein and it is also not published much.

I greatly appreciate your technical suggestions!