基本描述

规格或纯度

50 preps

英文名称

One Step Western Kit HRP (Mouse)

储存温度

2-8°C储存

运输条件

冰袋运输

产品介绍

产品内容：

Component	O665505 10 preps	O665505 50 preps
Blocking Buffer	100 ml	500 ml
Antibody Pretreat Solution（ HRP/Mouse ）	1 ml	5 × 1 ml
Dilution Buffer	100 ml	500 ml
Wash Buffer（ 10× )	100 ml	500 ml

产品简介：

一步法快速WB试剂盒（鼠）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 分钟，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 分钟）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鼠来源的实验系统使用。

注意事项：

1．客户自己准备鼠来源的一抗。

2．使用Blocking Buffer封闭液、Antibody Pretreat Solution（HRP/Mouse）抗体反应液（鼠）、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（鼠）需要通过预实验来确定最佳的稀释用量。

6．抗体反应液HRP（鼠）、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。特异性及亲和力不好的抗体建议不重复回收使用。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/Mouse）100 μl 到离心管中，加入鼠源一抗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（鼠）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/Mouse）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鼠单抗（CW0096）5 μg室温孵育40 min，洗膜后二抗羊抗鼠-HRP（CW0102）1：10000 稀释，室温4 0min，ECL（CW0049）曝光。

B: 一步法WB： beta-actin鼠单抗（CW0096）5 μg室温孵育 40 min，ECL（CW0049）曝光。

实例二抗原为E.coli多标签蛋白裂解液

C：普通WB对照：GST鼠单抗（CW0084）2.5 μg室温孵育 40 min，洗膜后二抗羊抗鼠-HRP（CW0102）1：10000 稀释，室温40 min，ECL（CW0049）曝光。

D：一步法WB： GST鼠单抗（CW0084）2.5ug 室温孵育 40min ， ECL（CW0049）曝光。

Product content：

Component	O665505 10 preps	O665505 50 preps
Blocking Buffer	100 ml	500 ml
Antibody Pretreat Solution（ HRP/Mouse ）	1 ml	5 × 1 ml
Dilution Buffer	100 ml	500 ml
Wash Buffer（ 10× )	100 ml	500 ml

Product Introduction:
The one-step rapid WB assay kit (mouse) is the latest Western Blot detection kit developed by Kangwei Century, which can obtain high-quality Western Blot results in about 1 hour. It is easy to operate, has high detection sensitivity, low background, does not require the addition of secondary antibodies, and has strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding, and secondary antibody binding) requires a long time, a complex experimental process, and requires multi-step condition optimization. After transferring the protein on the gel to the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier membrane with the primary antibody treated with antibody reaction solution. After washing three times (5 minutes each time), luminescence or colorimetric detection can be performed. This reagent kit is designed for use in experimental systems where the target protein primary antibody is derived from mice.

Notes:
1. The customer prepares their own mouse source primary antibody.
2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Mouse) antibody reaction solution (mouse), 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 (mouse) needs to be determined through preliminary experiments.
6. The antibody reaction solution HRP (mouse), 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. It is recommended not to reuse antibodies with poor specificity and affinity. 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/Mouse) 100 μ Add mouse 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 (mouse) 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/Mouse), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the surface of the membrane). Incubate at room temperature on a shaker at around 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.

Application examples:
Example 1 Antigen is 293T cell lysate
A: Normal WB control: beta actin mouse monoclonal antibody (CW0096) 5 μ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).
B: One step method WB: beta actin mouse monoclonal antibody (CW0096) 5 μ Incubate at room temperature for 40 minutes and expose ECL (CW0049).

Example 2 Antigen is E. coli multi label protein lysate
C: Normal WB control: GST mouse monoclonal antibody (CW0084) 2.5 μ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).
D: One step method WB: GST mouse monoclonal antibody (CW0084) was incubated at room temperature with 2.5ug for 40 minutes, and ECL (CW0049) was exposed.

安全和危险性（GHS）

SDS英文版

质量标准

质量标准

o665505_one step western kit hrp (mouse).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:

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

=

浓度

x

体积

x

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

x

体积 1 (V1)

=

浓度 2 (C2)

x

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

=

质量（小瓶）

÷

所需的复溶浓度

一步法快速WB（HRP）试剂盒（鼠）

库存信息

基本描述

安全和危险性（GHS）

质量标准

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器