I stained four tubes:
Tube 1: Unstained
Tube 2: Stained with only Total STAT3 labelled with PerCP cy 5.5.
Tube 3: Stained with only P-STAT3 (Y705) labelled with
Alexa flour 647.(APC Chanel )
Tube 4: MIX (Both the antibodies i.e T-STAT3 & pSTAT3).
The tube 2 which is only stained with Total STAT3 labelled with PerCP cy 5.5, In this tube there is a signal of APC (i.e. P-STAT3).
WHY there is signal of pSTAT3??
Because both are the different flurochromes?
2. In the MIX tube, stained with the both the antibodies i.e. (T-STAT3 & P-STAT3), then WHY there is only signal of only T-STAT3 which is perCP cy 5.5 labelled? but in the tube 2, stained with T-STAT3, both the signal are coming?
Kindly give your advice or suggestion.
Hello
Yes, i agree with Gulab ; should be work with the same protocol again and check on your antibodies and tubes label.
Good luck
Thank so much Gulab Fatima Rani & Saleh Alkarim.
I repeated this experiments so many times but every times results were same.
I acquired the all of these tubes in other flow Cytometer (Beckman Coulter),but results were similar.
What I do next????
Try using other mAb and maybe not Tandem mAb. Also use isotype controls and count the cells between washing step and Post-staining process.
Hi Adil
There is nothing wrong with your compensation controls in theory. However, notice that you are using an antibody against the phosphorilated form of STAT3 as a compensation control. If you don't have enough phosphorilation of STAT3 in your model (either because of cell activation status, phosphatase activity in your sample or other unknown reasons) you will not have a strong signal in that channel and therefore, you will not be able to compensate using that antibody.
I would recommend to separate both procedures.
For compensation:
You only need to keep your fluorochromes constant, not the actual antibodies. Compensation controls should have equal or higher MFI than your panels (never dimmer fluorescence). You can use an antibody directed against a different epitope as long as the antibody is conjugated to the same fluorochrome you will use in your panel. Consider also using compensation beads if you do not have a positive control.
For detection of phosphorilated proteins:
Choose carefully your positive controls. If using an antibody against a phosphorilated isoform, make sure you have the appropriate positive controls in your experimental design. Otherwise, you can never know if your negative staining is because of a bad staining, or because absence of phosphorilation. Beware of phosphatase activity in your sample, you might need to use an inhibitor to preserve pSTAT3 long enough to stain.
Finally... the antibody you are using, has been tested for FCM or is for WB? Remember in WB antibodies recognize linear peptides, whereas in FCM you need to recognize a native structure.
Hope this helps.
Andres
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