植物Western及IP细胞裂解液是采用一种非变性裂解方法来裂解细胞，并获得总蛋白的裂解液。所获得的蛋白质可以用于PAGE电泳，Western，免疫沉淀(ImmunolPrecipitation,IP)和免疫共沉淀(co-IP)等，本产品可以用于植物细胞、组织或原生质体样品，也可以用于动物的细胞或组织样品、真菌或细菌样品。本裂解液裂解的植物细胞、组织或原生质体样品，可以用于PAGE，Western，免疫沉淀(immunolprecipitation，IP)、免疫共沉淀(co-IP)和ELISA等。
植物Western及IP细胞裂解液主要由Tris、NaCl、低浓度TritonX-100，低浓度sodium pyrophosphate等组成，并含有多种蛋白酶抑制剂成分，可以有效抑制蛋白的降解，并维持原有的蛋白间相互作用。用本产品裂解液得到的蛋白，可以用BCA蛋白定量试剂盒测定蛋白浓度，由于含有较高浓度的TritonX-100等干扰物质，不宜用Bradford法测定由Western及IP细胞裂解液获得样本的蛋白浓度。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
操作步骤(仅供参考)：
将植物Western及IP细胞裂解液置于常温下溶解，混匀。取适量的裂解液，在使用前数分钟内加入PMSF(自备)，使PMSF的最终浓度为1mM，临用前配制，不可长期保存。
(一)对于培养的植物细胞样品：
1. 离心收集植物细胞，按照每50-100万细胞加入100-200微升裂解液的比例加入裂解液，用枪吹打数下，使裂解液和细胞充分接触，在冰上裂解2-10min。
2. 充分裂解后，10000-14000rpm离心3-5分钟，取上清，即可进行后续的PAGE、Western、免疫沉淀和免疫共沉淀等操作。
(二)对于植物原生质体样品：
1. 把组织剪切成细小的碎片，制备原生质体。
2. (可选)质粒转化原生质体，继续培养16-48小时，并根据需要给予适当的实验条件处理。
3. 100-500rpm低速离心收集原生质体。
4. 按照每50-100万原生质体加入100-200微升裂解液的比例加入裂解液。轻弹管底以充分混匀并裂解细胞。充分裂解后应没有明显的原生质体沉淀。如果原生质体量较多，必需分装成50-100万原生质体/管，然后再裂解。成团的50-100万原生质体较难裂解充分，而分散的50-100万原生质体由于裂解液容易和原生质体充分接触，相对比较容易裂解充分。
(三)对于植物组织样品：
1. 把植物组织剪切成细小的碎片。
2. 按照每20毫克植物组织加入100-200微升裂解液的比例加入裂解液。
3. 用玻璃匀浆器匀浆、研磨，直至充分裂解。也可以把组织样品冷冻后液氮研磨，研磨充分后再加入裂解液进行裂解。充分裂解后，10000-14000rpm离心3-5分钟，取上清，即可进行后续的PAGE、Western、免疫沉淀和免疫共沉淀等操作。
4. 如果植物组织样品本身非常细小和柔嫩，可以适当剪切后直接加入裂解液裂解，通过强烈vortex使样品裂解充分。充分裂解后，10000-14000rpm离心3-5分钟，取上清，即可进行后续的PAGE、Western、免疫沉淀和免疫共沉淀等操作。直接裂解的优点是比较方便，不必使用匀浆器或研磨设备，缺点是不如匀浆或研磨那样裂解比较充分。
注意事项：
1、去除贴壁细胞的培养液时，如果血清中的蛋白没有干扰，可以不用清洗。
2、如果裂解不充分可以适当增加裂解液的用量，如果需要高浓度的蛋白样品，可以适当减少裂解液的用量。
3、本产品低温存放可能产生少量絮状沉淀，可用水浴溶解，但应尽量缩短溶解时间，避免裂解液中的有效成分失效。产品首次溶解后可以分装小份，避免反复冻融。
4、细胞裂解的操作步骤，应置于冰上或4°C进行。
5、试剂开封后请尽快使用，以防影响后续实验效果。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Plant Western Blot and IP Cell Lysis Buffer employs a non-denaturing lysis method to lyse cells and obtain total proteins. The acquired proteins can be used for PAGE electrophoresis, Western blotting, Immunoprecipitation (IP), co-immunoprecipitation (co-IP), and other experiments. This product is applicable to plant cell, tissue, or protoplast samples, as well as animal cell or tissue samples, fungal, or bacterial samples. Plant cell, tissue, or protoplast samples lysed by this lysis buffer can be used for PAGE, Western blotting, Immunoprecipitation (IP), co-immunoprecipitation (co-IP), ELISA, and related applications.
Plant Western Blot and IP Cell Lysis Buffer mainly consists of Tris, NaCl, low-concentration Triton X-100, low-concentration sodium pyrophosphate, and other components. It also contains a variety of protease inhibitor ingredients, which can effectively inhibit protein degradation and maintain the original protein-protein interactions. The protein obtained using this lysis buffer can have its concentration determined by a BCA protein quantification kit. Due to the presence of interfering substances such as high-concentration Triton X-100, the Bradford method is not suitable for determining the protein concentration of samples obtained from Western Blot and IP Cell Lysis Buffer. This reagent is for research use only and not intended for clinical diagnosis or other purposes.
Operating Procedures (For Reference Only):
Thaw the Plant Western Blot and IP Cell Lysis Buffer at room temperature and mix well. Take an appropriate amount of the lysis buffer, add PMSF (prepared by the user) within a few minutes before use to adjust the final concentration of PMSF to 1 mM. The buffer with PMSF must be prepared immediately before use and cannot be stored for a long period.
(I) For Cultured Plant Cell Samples:
1. Collect plant cells by centrifugation. Add the lysis buffer at a ratio of 100–200 μL lysis buffer per 500,000–1,000,000 cells. Pipette gently several times to ensure full contact between the lysis buffer and cells, then lyse on ice for 2–10 minutes.
2. After complete lysis, centrifuge at 10,000–14,000 rpm for 3–5 minutes. Collect the supernatant, which can be used for subsequent experiments such as PAGE, Western blotting, immunoprecipitation (IP), and co-immunoprecipitation (co-IP).
(II) For Plant Protoplast Samples:
1. Cut the tissue into small pieces and prepare protoplasts.
2. (Optional) Transform protoplasts with plasmids, culture continuously for 16–48 hours, and treat with appropriate experimental conditions as needed.
3. Collect protoplasts by low-speed centrifugation at 100–500 rpm.
4. Add the lysis buffer at a ratio of 100–200 μL lysis buffer per 500,000–1,000,000 protoplasts. Flick the bottom of the tube gently to mix well and lyse the cells. After complete lysis, there should be no obvious protoplast precipitation. If the amount of protoplasts is large, they must be aliquoted into tubes with 500,000–1,000,000 protoplasts per tube before lysis. Aggregated protoplasts (500,000–1,000,000) are difficult to lyse completely, while dispersed protoplasts (500,000–1,000,000) are easier to lyse thoroughly because the lysis buffer can make full contact with them.
(III) For Plant Tissue Samples:
1. Cut the plant tissue into small pieces.
2. Add the lysis buffer at a ratio of 100–200 μL lysis buffer per 20 mg of plant tissue.
3. Homogenize and grind the tissue with a glass homogenizer until fully lysed. Alternatively, freeze the tissue sample, grind it in liquid nitrogen, and then add the lysis buffer for lysis after sufficient grinding. After complete lysis, centrifuge at 10,000–14,000 rpm for 3–5 minutes. Collect the supernatant, which can be used for subsequent experiments such as PAGE, Western blotting, IP, and co-IP.
4. If the plant tissue sample itself is very small and tender, it can be directly added to the lysis buffer after appropriate cutting, and lysed thoroughly by vigorous vortexing. After complete lysis, centrifuge at 10,000–14,000 rpm for 3–5 minutes. Collect the supernatant for subsequent experiments (PAGE, Western blotting, IP, co-IP, etc.). The advantage of direct lysis is convenience (no homogenizer or grinding equipment is needed), while the disadvantage is that it is less thorough compared to homogenization or grinding.
Precautions:
1. When removing the culture medium from adherent cells, washing is not necessary if proteins in the serum do not cause interference.
2. If lysis is insufficient, the amount of lysis buffer can be appropriately increased; if a high-concentration protein sample is required, the amount of lysis buffer can be appropriately reduced.
3. A small amount of flocculent precipitate may form when this product is stored at low temperatures. It can be dissolved in a water bath, but the dissolution time should be shortened as much as possible to prevent the active components in the lysis buffer from becoming ineffective. After the product is dissolved for the first time, it can be aliquoted into small portions to avoid repeated freeze-thaw cycles.
4. The operation steps for cell lysis should be performed on ice or at 4°C.
5. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
ZJ26F0130947
危险品化学品经营许可证（带存储）