基本描述

英文名称

One Step Western Kit HRP (Rabbit)

储存温度

2-8°C储存,禁止冻结

运输条件

冰袋运输

产品介绍

产品内容：

O665490	Component	50 T	Storage
O665490A	Blocking Buffer	500 mL	2-8℃. Do not freeze.
O665490B	Antibody Pretreat Solution (HRP/Rabbit)	5×1 mL	2-8℃. Do not freeze.
O665490C	Dilution Buffer	500 mL	2-8℃. Do not freeze.
O665490D	Wash Buffer (10×)	500 mL	2-8℃. Do not freeze.

产品简介：
一步法快速WB试剂盒（兔）是康为世纪最新研发的Western Blot检测试剂盒，能够在1小时左右得到高质量的Western Blot结果，且操作简便、检测灵敏度高、背景低、不需再加入二抗、系统稳定性强。常规的Western Blot间接法检测过程（封闭、一抗结合和二抗结合）需要较长的时间，实验流程复杂且需要多步条件优化。胶上的蛋白转移到载体膜上后，使用试剂盒中的封闭液孵育5 min，再用抗体反应液处理后的一抗孵育载体膜，经洗涤三次（每次5 min）后，即可进行发光或显色检测。本试剂盒针对目的蛋白一抗为兔来源的实验系统使用。

注意事项：

1. 客户需自己准备兔来源的一抗。

2. 使用Blocking Buffer封闭液、Antibody Pretreat Solution（HRP/Rabbit）抗体反应液（兔）、Wash Buffer（10×）漂洗液之前请充分混匀。

3. 漂洗液如在2-8℃保存时出现沉淀，请恢复到室温，把沉淀溶解后正常使用，1×漂洗液可在室温保存一个月。

4. 建议转膜完成后用丽春红等试剂染色，并把膜上多余部分剪去以增加试剂的使用效率。

5. 一抗和抗体反应液HRP（兔）需要通过预实验来确定最佳的稀释用量。

6. 抗体反应液HRP（兔）、抗体稀释液和抗体用量可根据膜的大小按比例放大或减少。

7. 加入一抗的抗体稀释液可以回收重复使用一次。特异性及亲和力低的抗体不建议重复使用。回收后抗体如在1-2天内使用放置在2-8℃，长期保存请在-20℃冻存，避免多次反复冻融。

8. 如果存在较高背景的情况，请调整抗体的用量，并增加洗膜次数。

9. 试剂盒内所有试剂请于2-8℃保存，避免冻融。

操作步骤：

本产品适用于转膜完成后的封闭及抗体孵育步骤，以5 cm×8 cm 膜为例：

1．漂洗液准备：取10 ml Wash Buffer（10×）用蒸馏水稀释至100 ml，即为1×Wash Buffer，待用。每次洗膜用8-10 ml。

2．封闭：转膜完成后，将膜浸没到10 ml Blocking Buffer中，室温封闭5分钟。

3．漂洗：倒掉封闭液，加入8-10 ml 1×Wash Buffer，于摇床上用较大速度漂洗1分钟。

4．洗膜的同时可准备抗体孵育液：取Antibody Pretreat Solution（HRP/Rabbit）100 μl 到离心管中，加入兔源一抗3-10 μg，枪头吸打至充分混匀，室温孵育5分钟。加入至10 ml Dilution Buffer中，充分混匀。注意：1）一抗的用量也可根据抗体的稀释度来进行调整。以抗体的最终稀释度1:1000为例，取100 μl 抗体反应液HRP（兔）到EP管中，加入10 μl一抗，加入到10 ml 抗体稀释液中，充分混匀，室温孵育 5分钟。 2）如果膜面积较小，可按比例减少抗体、反应液及稀释液的用量。

5．完成步骤3后，倒掉漂洗液，将一抗、Antibody Pretreat Solution（HRP/Rabbit）及Dilution Buffer混合而成的抗体孵育液加到膜上（确保孵育液完全浸没膜表面），在摇床上以60 rpm左右的速度室温孵育40分钟。

6．弃去（回收）抗体孵育液，用配制的1×Wash Buffer漂洗3-5次，每次3分钟。

7．进行后续检测。建议采用ECL或者DAB法进行检测。

应用实例：

实例一：抗原为293T细胞全裂解液

A：普通WB对照：beta-actin兔多抗（CW0097）3.3ug 室温孵育40min，洗膜后二抗羊抗兔-HRP（CW0103）1：10000 稀释，室温40min，ECL（CW0049）曝光

实例二抗原为293T细胞全裂解液

C：普通WB对照：PAK1，Epitomics兔单抗1：1000 室温孵育40min，洗膜后二抗羊抗兔-HRP（CW0103）1：10000 稀释，室温40min，ECL（CW0049）曝光

D：一步法WB： Epitomics兔单抗1：1000 室温孵育40min， ECL（CW0049）曝光。

Product content：

O665490	Component	50 T	Storage
O665490A	Blocking Buffer	500 mL	2-8℃. Do not freeze.
O665490B	Antibody Pretreat Solution (HRP/Rabbit)	5×1 mL	2-8℃. Do not freeze.
O665490C	Dilution Buffer	500 mL	2-8℃. Do not freeze.
O665490D	Wash Buffer (10×)	500 mL	2-8℃. Do not freeze.

Product Introduction：
The one-step rapid WB assay kit (rabbit) is the latest Western Blot detection kit developed by Kangwei Century, which can
Obtain high-quality Western Blot results within about 1 hour, with simple operation, high detection sensitivity, low background, and no
Additional secondary antibodies need to be added, with strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding)
Combining with secondary antibodies requires a long time, a complex experimental process, and requires multi-step optimization of conditions. The protein on the glue is transferred to
After coating the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier with the primary antibody treated with antibody reaction solution
After washing the membrane three times (5 minutes each time), it can undergo luminescence or color detection. This reagent kit is designed for target protein one
The use of an experimental system derived from rabbits.

Notes:
1. Customers need to prepare their own rabbit source primary antibody.
2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Rabbit) antibody reaction solution (rabbit), and Wash Buffer (10 x) rinse solution, please mix thoroughly.
3. If there is precipitation in the rinsing solution when stored at 2-8 ℃, please restore it to room temperature, dissolve the precipitation, and use it normally. The 1x rinsing solution can be stored at room temperature for one month.
4. It is recommended to stain the membrane with reagents such as spring red after the transfer is completed, and cut off any excess parts on the membrane to increase the efficiency of the reagents.
5. The optimal dilution amount for primary antibody and antibody reaction solution HRP (rabbit) needs to be determined through preliminary experiments.
6. Antibody reaction solution HRP (rabbit), antibody dilution solution, and antibody dosage can be increased or decreased proportionally according to the size of the membrane.
7. The antibody dilution solution containing the first antibody can be recycled and reused once. Antibodies with low specificity and affinity are not recommended for repeated use. If the recovered antibody is used within 1-2 days and stored at 2-8 ℃ for long-term storage, please freeze it at -20 ℃ to avoid repeated freeze-thaw cycles.
8. If there is a high background, please adjust the amount of antibodies and increase the number of times the film is washed.
9. All reagents in the reagent kit should be stored at 2-8 ℃ to avoid freezing and thawing.
Operation steps:
This product is suitable for the sealing and antibody incubation steps after membrane transfer, taking a 5 cm x 8 cm membrane as an example:
1. Preparation of rinsing solution: Dilute 10 ml of Wash Buffer (10 x) with distilled water to 100 ml, which is 1 x Wash Buffer. Set aside. Use 8-10 ml for each film wash.
2. Sealing: After the membrane transfer is completed, immerse the membrane in 10 ml Blocking Buffer and seal at room temperature for 5 minutes.
3. Rinse: Pour off the sealing solution, add 8-10 ml of 1 x Wash Buffer, and rinse at a high speed on a shaker for 1 minute.
4. Prepare antibody incubation solution while washing the membrane: Take Antibody Pretreat Solution (HRP/Rabbit) 100 μ Add rabbit derived primary antibody 3-10 into the centrifuge tube μ g. Suck and beat the gun head until thoroughly mixed, and incubate at room temperature for 5 minutes. Add to 10 ml Dilution Buffer and mix well. Note: 1) The dosage of primary antibody can also be adjusted according to the dilution of the antibody. Taking the final dilution of antibodies at 1:1000 as an example, take 100 μ Add HRP (rabbit) antibody reaction solution into the EP tube and add 10 μ Add the first antibody to 10 ml of antibody diluent, mix well, and incubate at room temperature for 5 minutes. 2) If the membrane area is small, the amount of antibodies, reaction solution, and diluent can be reduced proportionally.
5. After completing step 3, pour out the rinsing solution and add the antibody incubation solution mixed with primary antibody, Antibody Pretreat Solution (HRP/Rabbit), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the membrane surface). Incubate at room temperature on a shaker at a speed of about 60 rpm for 40 minutes.
6. Discard (recover) the antibody incubation solution and rinse 3-5 times with the prepared 1 x Wash Buffer, each time for 3 minutes.
7. Conduct subsequent testing. It is recommended to use ECL or DAB methods for testing.

Example 1: Antigen 293T cell lysate
A: Ordinary WB control: beta actin rabbit antibody (CW0097) 3.3ug incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed

Example 2 Antigen is 293T cell lysate
C: Ordinary WB control: PAK1, Epitomics rabbit monoclonal antibody 1:1000, incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed
D: One step WB: Epitomics rabbit monoclonal antibody was incubated at 1:1000 room temperature for 40 minutes, and ECL (CW0049) was exposed.

AI解读

安全和危险性（GHS）

SDS英文版

技术规格说明书

o665490_one step western kit hrp (rabbit).pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

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计算溶液所需的质量、体积或浓度。

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稀释计算器

计算配制储备溶液所需的稀释度。

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浓度 2 (C2)

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一步法快速WB（HRP）试剂盒（兔）

库存信息

基本描述

AI解读

安全和危险性（GHS）

技术规格说明书

质检证书（CoA,COO,BSE/TSE 和分析图谱)

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