Hello, I use the protocol to detect GSH and GSSG concenteration in HEK293T cell line which is treated with drug and the same DMSO as control ,I do have some interesting problems:
first, When I do the standard cuvre ,I realized that the signal of GSH is decrease with time increase but the signal of GSSG will increased as time increase, This make me confuse ,So I do GSSG standard curve again ,detect signal in 5min 10min 30min and 24hour ,Oh my god I saw the signaling at 26.4nM GSSG increase form 527(5min), 564(10min),621(30min),6977(24h), what makes me more confuse is that my blank control without GSSG but have 190ul KPE plus 10ul OPT also increase with time 71(5min), 89(10min),114(30min),4401(24h), Do you have meet this situation
second ,GSSG is hydrolysis in PH=12, but I dont know how much time it need to completely hydrolysis ,and OPT react with GSH is not influenced by the alkaline environment ,My point is that do I need incubate GSSG with NaOH for a span of time ,That When I detect signal as soon as I add GSSG NaOH and OPT, its 478(26.4nM ),but after incubate for 15min it become 523 .So I though maybe GSSG need preincubate with NaOH for a while.
The last one ,I do the same concentration of GSH and GSSG as protocol, but I get higher signal than the paper ,I read the signal as soon as I get all liquid in and shaking in 700rpm for 30s,and centrifuge for 20se in 3000rpm. I get 1378 in GSH at 26.4nM,and 484 GSSG at 26.4nM, much higher than yours ,Did there any problem ,What I want mentioned is that I get and good regress R^2=0.996 for GSH ,and 0.993 for GSSG,
So thankyou for your time !
10/17/2019 9:51:05 PM Reply