Hello!
I am trying to detect Cleaved caspase-3 by western blotting. Although I am able to my other protein of interest, but I cannot detect a cleaved form of caspase-3.
and am using 12.5 % gel and Antibody dilution is 1:500.
Does anyone have any tips how to detect active caspase-3 by WB or any suggestion what changes can I do.??
I think you need a good positive control. Protein extracts from cells treated with staurosporine worked for me.
You have to take into account the low molecular weight of cleaved caspase (around 13Kda), so I would rather use a 14-15% acrylamide gel. And if you have enough protein, 100ug per lane.
You can also check if you have a decrease in pro-caspase to see if your treatment is inducing caspase cleavage even if you can´t see the cleaved fragment.
Thank you for the suggestion Miguel A Zaballos
I will try with 15% acrylamide gel rather than 12.5%
and am using 50ug /lane and i think its enough because Loading control is fine my other protein of interest is fine except Cleaved caspase 3.
I am using #9661 antibody to detect Cleaved casp-3.
My protocol is-
1. Load 40 ug protein in a 15% gel.
2. primary Ab- 1:1000. O/N 4 degree
3. secondary Ab- 1:2000. 1 hr RT
4. wash extensively (30 min with 6-7 wash buffer changes)
5. 4-5 x-ray films and leave overnight.
You will have your band. Don't forget to add positive control here.
Thank you Rahul Kumar Sir, for your suggestion.
I would like to ask you what blocking buffer did you used for your experiment and how much percent BSA or Milk??
I am using 5% milk. then someone suggested to use 1% milk but didn't worked with both.
5% Milk is fine. I think 9661 antibody is very good, only thing is you will never get signals in 10-15 minutes exposure. I always put 4-5 films and leave overnight.
Good Luck
Maybe, your antibodies is not specific or maybe too old or does not work for any other reason.
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