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ELISA protocol 已有1人参与
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Step 1: Coat Capture Antibody (CA), 4ug/ml*100ul (720ug/ml*17.8ul, diluted with 1xPBS 3182.2ul), overnight. Step 2: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes. Step 3: Block Buffer (1% BSA in 1xPBS, 0.3g BSA in 30ml 1xPBS), 300ul/well, stand for 1h. Step 4: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes. Step 5: Standards or Samples, diluted with Reagent Diluent (2:1 dilute, 7 point, 1 Blank), 100ul/well, stand for 2h. Step 6: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes. Step 7: Detection Antibody (DA), 1.5ug/ml*100ul (270ug/ml*17.8ul, diluted with Reagent Diluent to 3182.2ul), 100ul/well, stand for 2h. Step 8: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes. Step 9: Sreptavidin-HRP, 1:200, diluted with Reagent Diluent (16ul in 3184ul), 100ul/well, stand for 20 min. Step 10: Wash Buffer, 300ul/well, stand for 3mins/time, *3 washes. Step 11: Glo A:B, 1:2, A 1066.7ul, B 2133.3ul), 100ul/well, stand for 5 min. Step 12: Read. |
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yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
- 散金: 2230
- 红花: 34
- 帖子: 2270
- 在线: 355.1小时
- 虫号: 2034994
- 注册: 2012-09-28
- 性别: MM
- 专业: 白血病
|
REAGENT PREPARATION Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. Working dilutions should be prepared and used immediately. A. Capture Antibody: Reconstitute with 1ml PBS, as recommended in the product datasheet. After reconstitution, store at 2 °C to 8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.* Dilute to the working concentration as recommended in the product datasheet. B. Biotinylated Detection Antibody: Reconstitute with 1ml Reagent Diluent, as recommended in the product datasheet. After reconstitution, store at 2 °C to 8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.* Dilute to the working concentration as recommended in the product datasheet. C. Standard: Reconstitute the standard with 0.5ml Reagent Diluent, as recommended in the product datasheet . Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Aliquot and store reconstituted standard at -70 °C. Refer to the product datasheet for the length of time the reconstituted standard may be stored. A seven point standard curve using 2-fold serial dilutions in Reagent Diluent. Refer to the product datasheet for the concentration of the high standard recommendation. D. Streptavidin-HRP (1 vial): 1.0 mL of streptavidin conjugated to horseradish-peroxidase. Store at 2 °C to 8 °C for up to 6 months after initial use.* DO NOT FREEZE. Dilute to the working concentration specified on the vial label using Reagent Diluent. Plate Preparation 1. Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) in PBS without carrier protein. Immediately coat a 96-well microplate with 100 µL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature. 2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 µL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels. 3. Block each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour. Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details. 4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition. |
2楼2013-10-09 04:19:18
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
- 散金: 2230
- 红花: 34
- 帖子: 2270
- 在线: 355.1小时
- 虫号: 2034994
- 注册: 2012-09-28
- 性别: MM
- 专业: 白血病
|
Standards curve is not accurate these days (duplicated wells had quite different results). Tests. Set: 1 2 3 4 A Standards-125pg/ml 125pg/ml 125pg/ml 125pg/ml B 125pg/ml 125pg/ml 125pg/ml 125pg/ml C 125pg/ml 125pg/ml 125pg/ml 125pg/ml D 125pg/ml 125pg/ml 125pg/ml 125pg/ml E 125pg/ml 125pg/ml 125pg/ml 125pg/ml F 125pg/ml 125pg/ml 125pg/ml 125pg/ml G 125pg/ml 125pg/ml 125pg/ml 125pg/ml H 125pg/ml blank air air Repeat 1 1 2 3 4 A 75420 104800 100610 171050 B 226310 89770 102150 127120 C 91280 85810 92720 133330 D 75140 97260 188420 108070 E 68740 93630 81660 123240 F 73220 118040 116920 134710 G 69340 102640 104190 141420 H 69360 36530 30 10 Repeat 2 1 2 3 4 A 72320 101410 97890 164680 B 227440 88230 98810 122710 C 91480 83770 90490 129730 D 74100 96000 182870 106370 E 67740 92070 79970 121070 F 70800 112810 113280 132400 G 66750 99510 101360 137430 H 68430 30920 20 20 Repeat 3 1 2 3 4 A 73510 100150 96550 161280 B 214830 85440 94580 120140 C 87440 82070 88510 128410 D 72690 94990 179210 104840 E 65640 89340 78720 118330 F 69700 111700 112570 128420 G 66150 96670 100200 134860 H 66430 32610 0 20 |
3楼2013-10-09 23:30:21
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4楼2014-08-05 08:46:36
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
- 散金: 2230
- 红花: 34
- 帖子: 2270
- 在线: 355.1小时
- 虫号: 2034994
- 注册: 2012-09-28
- 性别: MM
- 专业: 白血病
5楼2014-08-05 23:03:16
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
- 散金: 2230
- 红花: 34
- 帖子: 2270
- 在线: 355.1小时
- 虫号: 2034994
- 注册: 2012-09-28
- 性别: MM
- 专业: 白血病
6楼2014-08-05 23:04:25
