I am trying to detect endogenous Aurora B in the whole cell extract but I have high variability during the detection by LiCOR scanner. I use 2 different primary antibodies from BD technology and Cell signalling. Sometimes I detected AurB with BD antibodies and sometimes not (I am never been able to detect it with Cell signalling antibodies) but I didn't change the procedure. I hope somebody can help me..
Hi Mara, I hate to sound pessimistic but I read once that only about 20% of commercial antibodies actually work. Have both of these been used in publications? If not always run a positive controlling on your blots so you can confirm specificity of the bands. We have been using digital ECL imager and swear by Pierce westfemto ECL max detection.
I recommend you to read the enclose article. I hope will be helpful
BD and CST has good quality control compared to many other antibody companies. Since aurora kinases often localize to centrosomes, it could be a cell lysis issue. I have been using a lysis buffer which works for most of the proteins. You may try it. Lyse your cells as described in the following paper (also use the lysis buffer described in the paper below). This buffer is mild but efficient if you follow 40 minute lysis duration in ice with vortexing every 10 minutes. Also try to use primary antibodies at least (1:1000 dilution in milk) overnight incubation. Good Luck.
https://www.researchgate.net/publication/283306685_Smac_Mimetic_with_TNF-_Targets_Pim-1_Isoforms_and_Reactive_Oxygen_Species_Production_to_Abrogate_Transformation_from_Blebbishields
Article Smac Mimetic with TNF- Targets Pim-1 Isoforms and Reactive O...
I had problems with Sp1 antibody from Santa Cruz biotech.
In addition to above suggestions:
Are you working with polyclonal or monoclonal antibody?
If polyclonal.. then you should definitely see some signal with 1:200. Also make sure in what is the source of protein. We had once ordered an antibody that it was made against Hamster protein (it reacted with human, mouse and hamster according to the company's catalog..But) it did not work with our human cell extract!
If you do not get cross reactivity with the antibody that the company states( various conc starting from 1:200).. then there really is a problem. It is a good idea to see in their catalog on which cell extracts the antibody worked at what conc. Call the company and tell them about your situation.. they will ask you to send the western pics. Send them and ask if they can send the cell extracts to see if the problem is with antibody or your cell extract. Any good company will send it to you,. You can then perform western analyses with your cell lysate (25 and 50ug total protein), and company's cell lysate (25 and 50ug protein), and probe with various conc. (1:200, 1:500.. and 1:1000) of primary antibody. If there is a problem with antibody--> then the westerns with their cell extracts will not work either.
If monoclonal antibody, then look which epitope (protein sequence) the antibody is made against. If your protein is not denatured completely or if that particular epitope is hidden in the secondary structure of the protein, you may have this problem too.
One important thing..make sure that you also have western data on an endogenous control protein (anti-actin or anti-GAPDH) for each of these western blots. In future it will be useful to calculate the relative exp of your protein
Good Luck.
HI
Below antibody may useful.
https://www.thermofisher.com/order/genome-database/antibody/SIRT4-Antibody-Polyclonal/PA5-20965
all the best
just a few general thought.
you can check your protein in cell by immunostaining, its localization is quite specific to get info about the quality of your antibodies.
minor proteins in cell is much harder to detect than GAPDH, for example. i would get some positive control, transfected cells, or even rec protein (tagged even) to develop the analitical method.
CST antibody is polyclonal while BD antibody is monoclonal.
I tried 2 new protocols:
1. cell extract made directly in the loading buffer;
2. cell extract made by hypotonic buffer with protease and phosphatase inhibitors.
Thanks the first protocol I detected Aurora B but for studying protein degradation is not the best protocol because you can not quantify the proteins in your samples. And with the second protocol sometimes I was able to detect AurB and sometimes not.
Thank you for your suggestions. I will try some of these.
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