Hi. 1. Why do we use internal (negative ) control as actin?
2. Why we don't attach the detector enzyme directly to primary antibody?how secondary ab amplify the signal?
3. Why we need to separate proteins according to molecular weight first? Why not just add antibody with detector to bind to the protein?
i ask to FULLY digest the theory, not because I am not convinced.
Hi Heba
maybe I can help you with some of the questions :)
2. You can use the same conjugated secondary antibody to detect different primary antibodies. An anti-mouse secondary will bind to almost any mouse-sourced primary antibody, which allows some cost savings.
And several secondary antibodies will bind to the primary antibody resulting in an amplified signal.
3. For my study it is very important to distinguish between the native state, split products or sometimes isoforms of one protein which were all detected by my antibody. I can identify them by separate them according to their molecular weight.
Hi Heba
maybe I can help you with some of the questions :)
2. You can use the same conjugated secondary antibody to detect different primary antibodies. An anti-mouse secondary will bind to almost any mouse-sourced primary antibody, which allows some cost savings.
And several secondary antibodies will bind to the primary antibody resulting in an amplified signal.
3. For my study it is very important to distinguish between the native state, split products or sometimes isoforms of one protein which were all detected by my antibody. I can identify them by separate them according to their molecular weight.
1. B-Actin generally doesn't change under treatment conditions, so it's more of a control of how much protein is in each sample well. It's not always the best, as there are some situations in which the level of even control proteins can change as well. Alpha-tubulin is another option.
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