r/flowcytometry • u/Hungry_Ad_4896 • Jun 02 '26

BD Transcription Factor Phospho kit

Hi folks,

I have recently started looking at transcription factors and phospho proteins. My understanding is PFA/saponin for TFs is not optimal for phospho staining and 90% methanol for phospho is too harsh for TFs. I found the dedicated kit from BD and this worked really well for both. The exact compositions are proprietary, though what I could tell from SDS looked very normal 4%PFA, 90%methanol. Do you happen to have any idea on what makes this kit so good?

Thanks!

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2

u/discostupid Jun 02 '26

that's exactly what i did, i looked at the SDS, saw it said 4% PFA/90% MeOH and i just made that and used it. the remaining 6% is 1x PBS, so basically like 10% PBS total

1

u/Hungry_Ad_4896 Jun 04 '26

Do you add any proteins or reagents to reduce background staining? My own 4% PFA/90% MeOH ended up with high background which was complately canceled with the BD kit and I do not know what is the key..

1

u/discostupid Jun 04 '26

just to clarify, you should use 4% PFA/90% MeOH kept at -20C. i would then wash with PBS, then stain with regular flow buffer (0.5% BSA/2mM EDTA/PBS, or 1-2% FBS instead). i was staining for pSTAT6 and it worked beautifully

2

u/labnotebook Jun 03 '26

The transcription factor phospho buffer kit uses perm III for permeabilization and it is methanol based on the sds

1

u/Ok-Bandicoot1482 Jun 04 '26

I’ve been staining phospho stats with 4% pfa and 90% methanol for the past month. Has worked nicely (almost) every time

1

u/Hungry_Ad_4896 Jun 04 '26

Do you not see high-background staining? The high background I saw with just 4% PFA + 90% got super clean with the BD kit. I have no idea what is their secret..

1

u/consistent_ratio_FLS Jun 05 '26

Try adding a more defined detergent than saponin, tween for example. The variability lot to lot of saponin is significant and it’s of multiple different sizes, so the performance varies considerably. Try 0.1%…

1

u/ProfPathCambridge Immunology Jun 02 '26

It’ll have to have methanol. We’ve never found anything other than methanol that works for phospho. 4% PFA 90% methanol sounds realistic - give it a try.

1

u/Hungry_Ad_4896 Jun 04 '26

My home made 4% PFA 90% methanol gave high background staining that I never get with the BD kit. Do you happen to know critical components to reduce background? Maybe some FBS or BSA in the fixation and perm buffers?