24小时热门版块排行榜

返回列表

当前只显示满足指定条件的回帖，点击这里查看本话题的所有回帖

plantaqp

金虫 (正式写手)

应助: 14 (小学生)
贵宾: 0.012
金币: 738.3
散金: 774
红花: 9
帖子: 741
在线: 421.5小时
虫号: 293863
注册: 2006-11-05
专业: 植物生理与生化

[交流] 【求助/交流】我的Elisa 实验方法已有3人参与

ELISA
实验需要准备的buffer：
Buffer Carbonate-Bicarbonate 1M, pH 9.5 :
Dissolve 35g de Na2CO3 and 56g de NaHCO3 in 800ml water and  adjust to 1
liter

PBS 10X (for 1L):

KH2PO4 40 mM MW = 136.09 5.44 g/l
Na2HPO4 160 mM MW = 268.07 42.9 g/l
NaCl 1,15 M MW = 58.44    67.2 g/l
Adjust to  pH 7,3 with NaOH 6N

PBS-T :PBS 1X, Tween-20 0.1%

PBS-TB :
PBS 1X, BSA 1%, Tween-20 0.1%

ABTS :
250 mg in 500 ml citric acid anhydrous 0.1M, pH 4.35 (with NaOH) .
Aliquote by 10 ml et store  at  –20°C ;
For activity test  use 10ml ABTS, add 10 µl d’H2O2 30% and use in the following 20 minutes.

Solution STOP (= option)
80g SDS in 200ml H2O (40%) + 200 ml DMF (N,N-diméthyl formamide);
实验步骤：

 Load 100 ul antigen (PM at 0.02-5ng/ul or peptide at 0.5-25 pg/ul) (in buffer carbonate-bicarbonate 0.1M, pH 9.5) in wells of microtitration plaque Nunc “Maxi-Sorp Immuno Plate”.
 Incubate overnight at  4°C or 1-2h at 37°C ;
 Empty out the plate and rinse once with PBS 1X ;
 Saturate  sites with 200µl of PBS-TB for 30’ at 37°C ;
 Rinse 3 times with PBS-T ;
 Incubate with 100µl primary antibody  (1/2000 for AntiPIP2 and anti-PIP1 in PBS-TB) for 1-2 hours at  37°C ;
 Rinse 5 times with PBS-T ;
 Incubate with 100µl secondary antibody (1/2500 for Anti-rabbit HRP conjugated, in PBS-TB) for 1-2 h  à 37°C ;
 Rinse 5 times with PBS 1X ;
 Add 100µl de substrate (ABTS) and read at 416nm (or 405nm) après 2-30 min (program ELISA 405nm on VICTOR).

[ Last edited by plantaqp on 2010-7-14 at 22:22 ]

回复此楼