Caspase-3 Identification - (Feb/06/2007 )
Has anyone done caspase-3? If so, antibody from which compnay do you reccomend?
I have heard antibodies from Cell Signalling are not compatible with Licor Machine( Odyssey). I used Cell Signalling and dint get it.
Also, how do you determine if the cocncentration of the secondary and primary is the ideal. I know its trial and eroor. But what methods do you use to find out the optimum dilution of a primary and secondary? Look at previous papers or follow the antibody protocol of the company..
Thanks
I have heard antibodies from Cell Signalling are not compatible with Licor Machine( Odyssey). I used Cell Signalling and dint get it.
Also, how do you determine if the cocncentration of the secondary and primary is the ideal. I know its trial and eroor. But what methods do you use to find out the optimum dilution of a primary and secondary? Look at previous papers or follow the antibody protocol of the company..
Thanks
sorry for my lack of knowledge, but what is the Licor machine? by the way, we are pleased by Cell signalling Ab´s;
for secondary Ab in most cases a dilution of 1:1000 will do, or follow the manufacturer´s instructions; better vary the conc of the first Ab than of the secondary Ab
for secondary Ab in most cases a dilution of 1:1000 will do, or follow the manufacturer´s instructions; better vary the conc of the first Ab than of the secondary Ab
Its uses infrared Imaging to read memebranes. ANd its much sensitive than chemiluminescent detection. Thanks for the reply
I've used Cell Signalling's Ab to cleaved caspase-3 [#9661], works very well but there are two important points in getting it to work.
1] You must use the cell lysis buffer they specify [you can buy 10 ml of a 10x solution of this from Cell Signalling real cheap] - I compared this with my own regular lysis buffer and it made the difference between the blot not working at all and working very well.
2] You need load a LOT of cells per lane to detect cleaved caspase-3 even if using Cell Signalling's lysis buffer & protocol - I found ~10^6 cell per lane gave good result but even so [for ECL] you probably need expose for 5-10 minutes for optimum exposure.
Don't know anything about Licor but if it is way more sensitive than ECL you could get away with loading less on the gel though I would still reccomend point 1].