RIPA裂解液(强中弱套装)

阿拉丁生产的RIPA裂解液(RIPALysisBuffer)是一种传统的细胞组织快速裂解液。RIPA裂解液裂解得到的蛋白样品可以用于常规的Western、IP和ELISA等。RIPA的本意是Radio Immunoprecipitation Assay。RIPA裂解液的配方有很多种，根据其裂解液的强度大致可以分为强、中、弱三类。关于不同的RIPA裂解液以及阿拉丁生产的其它裂解液的主要特点和差异，以及如何选择裂解液可参考我们的相关网页：http://www.aladdin-e.com/ 。RIPA裂解液(强中弱套装)含有RIPA裂解液(强)、RIPA裂解液(中)和RIPA裂解液(弱)各50ml，便于实验时探索不同的实验条件。用RIPA裂解液裂解得到的蛋白样品，可以用阿拉丁生产的BCA蛋白浓度测定试剂盒测定蛋白浓度。由于含有较高浓度的去垢剂，不能用Bradford法测定由本裂解液裂解得到样品的蛋白浓度. 组分： 注意事项 ： 为取得最佳的使用效果，尽量避免过多的反复冻融。可以适当分装后使用。需自备PMSF。PMSF可以向阿拉丁订购。裂解样品的所有步骤都需在冰上或4℃进行。关于裂解液的选择，一方面可以参考我们的相关网页：http://www.aladdin-e.com/support/lysis-buffer.htm选择合适的裂解液；另一方面也需要通过一些预实验来摸索最佳的适合您实验条件的裂解液。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 对于培养细胞样品：1.融解RIPA裂解液，混匀。取适当量的裂解液，在使用前数分钟内加入PMSF，使PMSF的最终浓度为1mM。2.对于贴壁细胞：去除培养液，用PBS、生理盐水或无血清培养液洗一遍(如果血清中的蛋白没有干扰，可以不洗)。按照6孔板每孔加入150-250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。通常裂解液接触细胞1-2秒后，细胞就会被裂解。对于悬浮细胞：离心收集细胞，用手指把细胞用力弹散。按照6孔板每孔细胞加入150-250微升裂解液的比例加入裂解液。再用手指轻弹以充分裂解细胞。充分裂解后应没有明显的细胞沉淀。如果细胞量较多，必需分装成50-100万细胞/管，然后再裂解。3.充分裂解后，10000-14000g离心3-5分钟，取上清，即可进行后续的PAGE、Western和免疫沉淀等操作。裂解液用量说明：通常6孔板每孔细胞加入150微升裂解液已经足够，但如果细胞密度非常高可以适当加大裂解液的用量到200微升或250微升。每100万细胞用100微升RIPA裂解后获得的上清，其蛋白浓度约为2-4mg/ml，不同细胞有所不同。对于组织样品：1.把组织剪切成细小的碎片。2.融解RIPA裂解液，混匀。取适当量的裂解液，在使用前数分钟内加入PMSF，使PMSF的最终浓度为1mM。3.按照每20毫克组织加入150-250微升裂解液的比例加入裂解液。(如果裂解不充分可以适当添加更多的裂解液，如果需要高浓度的蛋白样品，可以适当减少裂解液的用量。)4.用玻璃匀浆器匀浆，直至充分裂解。5.充分裂解后，10000-14000g离心3-5分钟，取上清，即可进行后续的PAGE、Western和免疫沉淀等操作。每20mg冻存的小鼠肝脏组织用200微升RIPA裂解液裂解后获得的上清，其蛋白浓度约为15-25mg/ml，不同状态的不同组织有所不同。6.如果组织样品本身非常细小，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。然后同样离心取上清，用于后续实验。直接裂解的优点是比较方便，不必使用匀浆器，缺点是不如使用匀浆器那样裂解得比较充分。注：RIPA裂解液的裂解产物中经常会出现一小团透明胶状物，属正常现象。该透明胶状物为含有基因组DNA等的复合物。在不检测和基因组DNA结合特别紧密的蛋白的情况下，可以直接离心取上清用于后续实验；如果需要检测和基因组结合特别紧密的蛋白，则可以通过超声处理打碎打散该透明胶状物，随后离心取上清用于后续实验。如果检测一些常见的转录因子，例如NF-kappaB、p53等时，通常不必进行超声处理，就可以检测到这些转录因子。附录：阿拉丁生产的各种裂解液主要特点、差异和选择首先请参考下表，了解各种裂解液的主要特点和差异。产品编号P0013P0013BP0013CP0013DP0013FP0013GP0013JP0013K产品名称Western及IP细胞裂解液 RIPA裂解液(强) RIPA裂解液(中) RIPA裂解液(弱) NP-40裂解液 SDS裂解液 Western及IP细胞裂解液(无抑制剂) RIPA裂解液(强，无抑制剂) 有效裂解成分1% Triton X-1001% Triton X-100, 1% deoxycholate, 0.1% SDS1% NP-40, 0.5% deoxycholate, 0.1% SDS1% NP-40, 0.25% deoxycholate1% NP-401% SDS1% Triton X-1001% Triton X-100, 1% deoxycholate, 0.1% SDS裂解强度温和强中温和温和强温和强对膜蛋白的提取一般很好较好一般一般很好一般很好对胞浆蛋白的提取很好很好很好很好很好很好很好很好对核蛋白的提取较好很好较好较好较好很好较好很好胞浆磷酸化蛋白提取很好很好很好很好很好很好很好很好细胞核转录因子提取很好很好很好很好很好很好很好很好含蛋白酶抑制剂是是是是是是否否含磷酸酯酶抑制剂是是是是是是否否主要用途WB, IP,co-IPWB, IPWB, IPWB, IP, co-IPWB, IP,co-IPWB, ChIPWB, IP,co-IPWB, IP用于普通的Western、IP或co-IP，我们推荐使用Western及IP细胞裂解液，该裂解液已被国内各大研究机构广泛使用，发表大量SCI论文，用户普遍反映很好。裂解细胞或组织后，没有非常粘滞的透明状DNA团块形成，不必采用超声处理等就可以非常理想地用于后续操作。另外该裂解液裂解的产物也适合用于磷酸化蛋白的Western检测。 对于某些特殊蛋白的IP，如果发现Western及IP细胞裂解液效果不是非常理想，可以尝试用RIPA裂解液(强、中或弱)或NP-40裂解液。如果发现IP的时候背景很高，即非特异的蛋白也被IP下来，则需要选用裂解强度较高的裂解液，例如RIPA裂解液(强或中)。如果发现目的蛋白无法被IP下来，则说明裂解液的强度过强，可以使用较为温和的裂解液例如RIPA裂解液(弱)或NP-40裂解液。对于某些难溶解蛋白的Western，如果发现Western及IP细胞裂解液效果不是非常理想，可以尝试使用裂解强度更高的裂解液例如RIPA裂解液(强、中)或SDS裂解液。使用RIPA裂解液(强)的用户也非常多，发表了大量SCI论文。用于特定用途需要自行添加特定抑制剂或不需要添加抑制剂时，可以考虑选购P0013J或P0013K。P0013J在很多时候可以兼容酶活性和生物小分子的检测，对于特定的酶或生物小分子的检测是否兼容需 要自行测试，阿拉丁不提供具体的应用信息。P0013J的裂解能力比P0013K弱一些，但用于酶活性和生物小分子时，P0013J的兼容性通常会更好一些。 Aladdin's RIPA Lysis Buffer is a traditional lysis buffer for rapid cell and tissue lysis. The obtained sample lysate can be used for routine PAGE, Western, IP, Co-IP, and ELISA analysis.This product can be used to lyse animal, plant, bacteria and yeast samples.RIPA is the abbreviation of Radio Immunoprecipitation Assay. Based on the lysis capability of RIPA Lysis Buffers, they are divided into strong, medium and weak lysis buffers. For the main characteristics and comparison of Aladdin's RIPA Lysis Buffer and other lysis buffers, please visit the website at https Precautions ： For the best performance, this product should be avoided repeated freeze-thaw. Store aliquots at -20℃.PMSF (, ST506) is required, but not supplied in this product. Inhibitor cocktails can also be used for better lysis results. provides a variety of inhibitor cocktails, such as Protease and Phosphatase Inhibitor Cocktail for General Use , Protease and Phosphatase Inhibitor Cocktail for General Use, MS-safe , Protease and Phosphatase Inhibitor Cocktail for Mammals , Protease and Phosphatase Inhibitor Cocktail for plant , Protease and Phosphatase Inhibitor Cocktail for Fungi , and Protease and Phosphatase Inhibitor Cocktail for Bacteria . If phosphorylated proteins are not to be detected, inhibitors of protease only can be used.All procedures for sample lysis should be performed on ice or at 4℃.An appropriate sample lysis buffer can be selected by referring to the website at https://www.aladdin-e.com or optimized by preliminary tests.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1. Lysis of cultured cellsa. Thaw RIPA Lysis Buffer completely and mix well. Just before use, take an appropriate amount of RIPA Lysis Buffer and add PMSF to a final concentration of 1mM. Other protease and phosphatase inhibitors can also be used if required.b. For adherent cells: Aspirate the culture medium and wash cells once with PBS, buffered saline or serum-free culture medium to remove protein residues that may interfere with cell lysis. Add 150-250μl of lysis buffer for each well of cells in 6-well plates and pipette up and down several times to ensure full contact of cells with the lysis buffer. Generally, it takes 1-2 sec to lyse animal cells or 2-10 min to lyse plant cells.For suspension cells: Collect cells by centrifugation, vortex gently or flick the bottom of the tube to disperse cells as much as possible. Add 150-250μl of lysis buffer for each well of cells in 6-well plates, flick the bottom of the tube to resuspend and lyse cells thoroughly. There should be no obvious precipitates after full lysis. For large amounts of cells, dispense them into 0.5-1.0×106 cells per tube for lysis. For bacteria or yeasts: Collect bacteria or yeasts by centrifuging 1ml of cultures. Aspirate the supernatant and wash the pellet once with PBS if necessary. After removing the supernatant, vortex gently or flick the bottom of the tube to disperse cells, and then add 150-250μl of lysis buffer. After resuspending cells completely in the lysis buffer, lyse cells on ice for 2-10 min. For better lysis, bacteria or yeasts can be treated with lysozyme or lyticase before adding the RIPA Lysis Buffer.Note: Generally, for one well of cells in 6-well plates or 1ml of bacterial or yeast cultures, 150μl of lysis buffer is sufficient. For higher cell densities, use 200-250μl of lysis buffer. When lysing 1 million animal cells with 100μl of lysis buffer, the protein concentration of the lysate is approximately 2-4 mg/ml, which is dependent on the type of cells. c. After full lysis of cells, centrifuge at 10,000-14,000×g for 3-5 minutes and take the supernatant for PAGE, Western or IP analysis. 2. Lysis of tissuesa. Cut tissue into small pieces.b. Thaw the RIPA Lysis Buffer completely and mix well. Just before use, take an appropriate amount of RIPA Lysis Buffer and add PMSF to a final concentration of 1mM. Other protease and phosphatase inhibitors can also be used if required.c. Add 150-250μl of lysis buffer per 20mg of tissues. The amount of lysis buffer can be adjusted based on the lysis results.d. Homogenize tissues thoroughly with a glass homogenizer or 's TissueMasterTM Handheld Homogenizer . e. Centrifuge at 10,000-14,000×g for 3-5 minutes and take the supernatant for PAGE, Western or IP analysis. When lysing 20mg of mouse liver tissue with 200μl of lysis buffer, the protein concentration of the lysate is approximately 15-25 mg/ml, which is dependent on the state of tissues.f. For tiny tissues, add lysis buffer after appropriate cutting and lyse tissues with vigorous vortex. Centrifuge and take the supernatant for subsequent analysis. This lysis method is convenient, with no homogenization needed, but the lysis result is not as thorough as that from the homogenization method.Note: It is normal to see a small clump of transparent gelatinous substances containing genomic DNA in RIPA cell lysate. In cases where DNA-binding proteins will not be analyzed, the supernatant can be analyzed directly. Otherwise, break the gelatinous substance with an ultrasonicator and then analyze the supernatant after centrifugation. Some common transcription factors such as NF-kappaB and p53 can be detected in the supernatant without ultrasonic processing.Appendix：Comparison and selection of Lysis Buffers from : Cat. No. Component QuantityP0013P0013BP0013CP0013DP0013FP0013GP0013JP0013KProduct NameLysis Buffer for Western&IPRIPA Lysis Buffer RIPA Lysis Buffer (Medium)RIPA Lysis Buffer (Weak)NP-40 Lysis BufferSDS Lysis BufferLysis Buffer for Western&IP (No Inhibitors)RIPA Lysis Buffer Effective Component1% Triton X-1001% Triton X-100, 1% deoxycholate, 0.1% SDS1% NP-40, 0.5% deoxycholate, 0.1% SDS1% NP-40, 0.5% deoxycholate 1% NP-401% SDS1% Triton X-1001% Triton X-100, 1% deoxycholate, 0.1% SDSLysis StrengthMildStrongMediumMildMildStrongMildStrongExtraction of Membrane ProteinsFairExcellentGoodFairFairExcellentFairExcellentExtraction of Cytosolic ProteinsExcellentExcellentExcellentExcellentExcellentExcellentExcellentExcellentExtraction of Nuclear ProteinsGoodExcellentGoodGoodGoodExcellentGoodExcellentExtraction of Phosphorylated Proteins in CytosolExcellentExcellentExcellentExcellentExcellentExcellentExcellentExcellentExtraction of Transcription Factors in NucleusExcellentExcellentExcellentExcellentExcellentExcellentExcellentExcellentProtease InhibitorsYesYesYesYesYesYesNoNoPhosphatase InhibitorsYesYesYesYesYesYesNoNoSpecies CompatibilityHighHighHighHighHighHighHighHighApplicationsWB, IP, Co-IPWB, IPWB, IPWB, IP, Co-IPWB, IP, Co-IPWB, ChIPWB, IP, Co-IPWB, IPFor general Western, IP or co-IP assays, we recommend using Lysis Buffer for Western & IP (, P0013) which has been extensively used with excellent performance. Lysing cells with this lysis buffer does not need ultrasonic processing. The obtained sample lysate has no gelatinous DNA clump and is ideal for subsequent studies as well as detection of phosphorylated proteins by Western.For IP of some special proteins, RIPA Lysis Buffer or NP-40 Lysis Buffer can be attempted when Lysis Buffer for Western & IP (, P0013) does not work well. If the background of IP result is high, a stronger lysis buffer such as RIPA Lysis Buffer should be used. If the target protein can not be immunoprecipitated, it indicates that the lysis buffer used is too strong, and a milder lysis buffer such as RIPA Lysis Buffer (Weak) and NP-40 Lysis Buffer should be tried.For Western blot of some proteins hard to be soluble, RIPA Lysis Buffer or SDS Lysis Buffer can be attempted when Lysis Buffer for Western & IP (, P0013) does not work well. RIPA Lysis Buffer is also extensively used by investigators and has been cited in large amounts of articles.For special applications where no inhibitors or a particular inhibitor is needed, P0013J or P0013K can be selected. P0013J is compatible with enzymatic activity assay and small molecule detection in most cases, but for special enzymes or small molecules, users should check the compatibility on their own. P0013J has weaker lysis capability than P0013K, but P0013J generally has better compatibility with enzyme and small molecule studies.

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