The kit which you mentioned is the HRP based detection kit for Western blot. You can very well use any HRP conjugated secondary antibody for use with that kit.

We used rabbit primary antibodies from Sigma and Santa Cruz for western blotting and immunofluorescence. The resulting signal was very good. But we used luminiscent, not chorogenic detection. And if you will face some problems - it's a commercial kit, sent your questions to manufacturer.

Kit is good for start but I will recommend, you should order antibodies which is well reported in literature for your protein of interest and then you can order secondary-HRP antibody from santacruz. Then you will be needing ECL detection system, X-Ray film and a dark room to develop it. As far as my experience, I had a good experience with rabbit antibodies and they work well from cell signaling, santacruz, Millipore, abacam.

I don't see any problems with rabbit antibodies. If the antibody is good (not all are) then it should work. We use antibodies from different species, depending on availability. And it opens the possibility to use different primary antibodies at the same time.

Hey I have used primary antibody developed in rabbit from different companies. And I have used for western, immunofluorescence, both worked well. Except some of the cases most of the time they worked just well with me... And ya I have also used HRP conjugated secondary antibody from Santacruz. I think its the specific problem with your primary antibody and it doesnt involve whether its raised in rabbit of mouse....

Hi, we use rabbit primary antibodies from Abcam for western blotting. Our results are clear and good. But our detection method is luminiscent.

Actually, this is my first trial to get involved in western blot. Thanks to all of the distinguished scientists. :)

You need to optimize the concentration of your primary and as well as that your secondary antibody. Therefore run the SDS-Gel with a single slot (over the whole width of your gel), transfer the proteins (the best version is an overnight tank blot at 15 °C [beware: SDS will crystallize below 15 °C]) on a single membrane sheet. Block the membrane, cut the membrane into stripes (each about 3 mm), incubate them with different concentrations of your primary antibody (an optimal concentration is usually below 1 µg/mL in PBS 0,3 mol/L NaCl, 0.1& v/v Tween 20). The increased salt concentration will decrease your background by avoiding unspecific reactions, the tween as a detergent will avoid unspecific hydrophobic reactions.

Wash the stripes and incubate them with different concentrations of your labeled antibody. The substrate reaction should take at least 10 - 20 minutes to keep the opportunity for a working rhythm.

So you have three points to optimize your reaction scheme. First antibody incubation, second and substrate reaction. Make sure that the substrate reaction is stopped completely by a sufficient washing (simple 4 x 2 - 5 min with water).

No problem at all. I've used primary Ab raised in rabbit (Sigma) for detection of stress protein. Result of your blot depends on the reactivity of your protein with the antibody not with the species in which the Ab is raised. So go ahead.

No problem at all. I've used primary Ab raised in rabbit (Sigma) for detection of stress protein. Result of your blot depends on the reactivity of your protein with the antibody not with the species in which the Ab is raised. So go ahead.

Same as others. Rabbit antibodies are OK as far as the protein to be recognized is not a rabbit protein. In my hands, it is better than the animal source of the primary and second antibodies are different, but the important point is the quality of the antibodies and the appropriate dilution. Santa cruz are usually very good. Good luck.

I have routinely used rabbit antibodies for immunoblotting cell/tissue lysates and they work well. For some proteins, there are multiple bands but you can minimize this by increasing the duration and/or stringency of the washes and by ensuring that you are applying a high quality lysate to your gel. For the best results, it is very important to handle the lysates carefully during their preparation, by keeping them on ice at all times, by ensuring that an appropriate mixture of fresh protease inhibitors are used in the lysis buffer and by completing the entire procedure in less than 2 hours. Lysates should be boiled with PAGE sample buffer and frozen immediately, if they are not used right away. For many proteins it is best to use the lysates within a week, rather than storing them for an extended period of time, even at -80C. Background noise is not generally a problem with rabbit antibodies. If you have a problem with low signal intensity, you may want to think about using chemiluminescent detection for increased sensitivity. I can reliably detect, with good specificity, even proteins that have low abundance (at least in some cell lines), such as carboxypeptidase D, EDD and some hormone receptors. For the secondary, I recommend using donkey anti-rabbit-HRP from Amersham or goat anti-rabbit-HRP from Sigma or Abcam. All three can be used at 1:5000 for most proteins, when using chemiluminescent detection.

There is no problem with using rabbit antibody per se, as a few people have pointed out already. The problems you listed could happen to antibodies raised in any other animals.

I agree with everybody. I have been working with many primary antibodies from many animals and the problems we have was the same you listed. I have much problems with goat antibodies already.

I agree with everybody. I have been working with many primary antibodies from many animals and the problems we have was the same you listed. I have much problems with goat antibodies already.