Hi all,
Does anyone have experience with detecting _intracellular_ myc using FACS (ie not surface-bound)? (We want to quantify the % of cells in culture that contain a myc-fusion protein). Clone 9E10 for example? If anyone has done this, which permeabilisation protocol would you recommend?
Thank you for your time,
Best,
Josephine
I don't have my protocol here with me but I found a protocol very similar with what I did. Just be careful with vortexing.
A fast and standardized protocol for intracytoplasmic immunofluorescence provides a fixation with PFA, time are variable depending on cell type, for example lymphocytes requires only 5 minutes at 4C°. For other cell type such as MSC almost ten minutes are necessary in order to allow a good fixation state. The final PFA concentration is 1%.
Then you need to permeabilize, you can use Triton X-100,2% final concentration. Also in this case 5 minutes are enough, always 4C°. After that wash with PBS alwas in refrigered centrifuge and stain the samples with the antibody.
If you need to detect antigen that are usually secreted such as cytokines and so one, you need, preventively (24 hours before the IF protocolo) to add the GolgiStop or other Protein Transport Inhibitor, in order to avoid the release in the culture medium..The day after harvest your cells wash and stain as aforementioned.
Hi Andrea,
Thank you for your response!
You don't have experience in particular with any myc antibody using this protocol?
/Josephine
Hi Josephine,
my pleasure. Unfortunately nope.. Usually I applied it for FoxP3, on T-Reg cells, or to detect cytokines. I think that the protocol aforementioned can fit without any type of problem..I just saw that you use an indirect staining so maybe you can try to increase your signal by using a secondary Ab conjugated with a very strong fluorochrome such as APC... It also depend by if you need to detect the intracytoplasmic myc or the TF inside the nuclei..
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