m*********e
发帖数: 378 | 1 Dear all,
I have some problem with the antibodies from cellsignal for mTOR, p-mTOR (
2448), Rictor, Raptor and p-AKT(308). If anyone have used some good
antibodies for these protein please tell me the company and catlog number.
Thanks a lot. | s********n
发帖数: 248 | 2 我用过cell signaling 的pAKT T308,这个克隆是最好的
http://www.cellsignal.com/products/2965.html
block 用5%milk/PBST (0.02% TWEEN 20), dilute primary antibody in either 5%
milk or 5%BSA/PBST, 4 degree overnight.
quick wash 2/3 times, then wash 3 times*5 min with PBST,
dilute 2nd antibody (JACKSON LAB HRP-GOAT anti-Rabbit light chain) 1:2500 in 5%MILK/PBST
(如果杂带多，需要把二抗稀释在5%milk里), RT for 1h; 如果杂带不多，可以稀释1:
15000 in PBST, RT for 45min。
quick wash 2/3 times, then wash 5 times*5 min with PBST.
【在 m*********e 的大作中提到】
: Dear all,
: I have some problem with the antibodies from cellsignal for mTOR, p-mTOR (
: 2448), Rictor, Raptor and p-AKT(308). If anyone have used some good
: antibodies for these protein please tell me the company and catlog number.
: Thanks a lot.
| m*********e
发帖数: 378 | 3 THANKS SO MUCH!!!!!
^o^
in 5%MILK/PBST
【在 s********n 的大作中提到】
: 我用过cell signaling 的pAKT T308,这个克隆是最好的
: http://www.cellsignal.com/products/2965.html
: block 用5%milk/PBST (0.02% TWEEN 20), dilute primary antibody in either 5%
: milk or 5%BSA/PBST, 4 degree overnight.
: quick wash 2/3 times, then wash 3 times*5 min with PBST,
: dilute 2nd antibody (JACKSON LAB HRP-GOAT anti-Rabbit light chain) 1:2500 in 5%MILK/PBST
: (如果杂带多，需要把二抗稀释在5%milk里), RT for 1h; 如果杂带不多，可以稀释1:
: 15000 in PBST, RT for 45min。
: quick wash 2/3 times, then wash 5 times*5 min with PBST.
| m*********e
发帖数: 378 | 4 还想问一下 你是用什么显色的，ECL, ECL plus,还是pierce 的pico,duro什么的
in 5%MILK/PBST
【在 s********n 的大作中提到】
: 我用过cell signaling 的pAKT T308,这个克隆是最好的
: http://www.cellsignal.com/products/2965.html
: block 用5%milk/PBST (0.02% TWEEN 20), dilute primary antibody in either 5%
: milk or 5%BSA/PBST, 4 degree overnight.
: quick wash 2/3 times, then wash 3 times*5 min with PBST,
: dilute 2nd antibody (JACKSON LAB HRP-GOAT anti-Rabbit light chain) 1:2500 in 5%MILK/PBST
: (如果杂带多，需要把二抗稀释在5%milk里), RT for 1h; 如果杂带不多，可以稀释1:
: 15000 in PBST, RT for 45min。
: quick wash 2/3 times, then wash 5 times*5 min with PBST.
| n***1
发帖数: 45 | 5 这些抗体都是CELL SIGNALING的好用，正常应该都没问题
【在 m*********e 的大作中提到】
: Dear all,
: I have some problem with the antibodies from cellsignal for mTOR, p-mTOR (
: 2448), Rictor, Raptor and p-AKT(308). If anyone have used some good
: antibodies for these protein please tell me the company and catlog number.
: Thanks a lot.
| s********n
发帖数: 248 | 6 ECL,take the ECL out when you are done with 2nd antibody, prewarm in room temp for about 25min, then use 4ml(buffer A)100ul(buffer B) for a 6*4cm^2 membrane, 4min, then develop.
【在 m*********e 的大作中提到】
: 还想问一下 你是用什么显色的，ECL, ECL plus,还是pierce 的pico,duro什么的
:
: in 5%MILK/PBST
| m*********e
发帖数: 378 | 7 Maybe we are using antibodies for the same protein with different catalog
numbers. Will you please let me know the ones you are using? I just think
it is not the problem of my technique because I tested many antibodies from
Cellsignal and they work pretty good.
Thanks a lot !
【在 n***1 的大作中提到】
: 这些抗体都是CELL SIGNALING的好用，正常应该都没问题
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