Aladdin RIPA cracking solution (strong) - readily available in stock

Aladdin R301899-100ml RIPACracking solution (strong)

Aladdin RIPA cracking solution (strong) - readily available in stock

Product Item Number:R301899-100ml

Product Name:RIPACracking solution (strong)

Product Specifications100ml

Product Introduction:

Specification or purity: No inhibitor

English name:RIPA Lysis Buffered Solution(Strong)

Storage temperature:2-8°Cstore

Transportation conditions: Ice pack transportation

RIPALysis is a traditional tissue and cell lysis buffer, mainly used to extract soluble proteins from animal tissues and mammalian cells, and can be used to lyse adherent cells and suspended cells. According to the strength of its cracking solution, it can be divided into three categories: strong, medium, and weak, and corresponding products can be selected according to experimental needs.RIPAlysis buffer(strong)It can effectively extract nuclear, membrane, and cytoplasmic proteins, and the extracted proteins can be applied to protein quantificationWestern BlotTheIPWaiting for testing and analysis.

The specific product parameters are subject to the parameters in the product manual received

Aladdin RIPA cracking solution (strong) - readily available in stock

Usage

(one)Adherent culture of cells

1.Dissolve and mix the product at room temperature, and add enzyme inhibitors according to experimental requirements.

2.Remove the culture medium of adherent cells and usePBSTheNSOr clean with serum-free culture medium1Next, centrifuge at low speed, discard the supernatant, and retain the precipitate.

3.according to 6 Add to each hole of the orifice plate150～250μlAdd the proportion of lysis buffer containing enzyme inhibitors to this product. Gently blow the pipette to ensure full contact between the lysate and the cells. Placed on ice or4℃Lysis, usually the lysate acts on cells 1～3sInside, the cells will be lysed. usually 6 Add cells to each well of the well plate150μlThe lysis buffer is sufficient, but if the cell density is very high, the amount of lysis buffer can be appropriately increased 200～250μl.

4.10000～12000g，4℃centrifugation 5～10min(If there is no low-temperature centrifuge, centrifugation at room temperature is also possible)Take the supernatant.

5.subsequentSDS-PAGETheWesternOperations such as immunoprecipitation and immunoprecipitation.

(two)Suspension cultured cells

1.Dissolve and mix the product at room temperature, then add enzyme inhibitor.

2.Suspend cells by low-speed centrifugation, discard supernatant, and collect sediment.

3.Gently tap the cells with your fingers to loosen them. according to 6 Add cells to each well of the well plate150～250μlAdd the proportion of lysis buffer containing enzyme inhibitors to this product. usually 6 Add cells to each well of the well plate150μlThe lysis buffer is sufficient, but if the cell density is very high, the amount of lysis buffer can be appropriately increased 200～250μlGently tap with your fingers to fully lyse the cells, and after thorough lysis, there should be no obvious fine or cellular precipitates.

4.10000～12000g，4℃centrifugation5～10min(If there is no low-temperature centrifuge, centrifugation at room temperature is also possible)Take the supernatant.

5.Carry out subsequent actionsSDS-PAGETheWesternOperations such as immunoprecipitation and immunoprecipitation.

(three)Organizational samples

1.Dissolve and mix the product at room temperature, then add enzyme inhibitor.

2.Group upzhiCut into small fragments, the smaller the better.

3.Freeze in liquid nitrogen or ultra-low temperature freezer30minThe above organizations should be quickly ground with liquid nitrogen, and the grinding process should be controlled as much as possible1～2minInternally, to reduce protein degradation.

4.According to each20mgJoin the organization 150～250μlThe ratio of lysis buffer is to add lysis buffer containing enzyme inhibitors. On ice or4℃cleavage 15～30min.

5.step 3、4 The following process can also be adopted: according to each20mgJoin the organization 150～250μlAdd this product containing enzyme inhibitors to the ratio of the lysate. Use a glass homogenizer or tissue grinder to homogenize until fully lysed, and try to control the process as much as possible 1～2minInternally, to reduce protein degradation.

6.10000～12000g，4℃centrifugation 5～10min(If there is no low-temperature centrifuge, centrifugation at room temperature is also possible)Take the supernatant.

7.Carry out subsequent actionsPAGETheWesternOperations such as immunoprecipitation and immunoprecipitation.

Precautions

1.After removing the culture medium of adherent cells, if the proteins in the serum do not interfere, cleaning is not necessary.

2.If the lysis is not sufficient, the amount of lysis buffer can be appropriately increased. If high concentration protein samples are required, the amount of lysis buffer can be appropriately reduced.

3.If there are a large number of cells, they must be divided into 50～100 Ten thousand cells/Centrifuge the tube and then crack it. Large clusters of cells are difficult to fully lyse, while a small number of cells are relatively easy to fully lyse due to the easy contact between the lysate and the cells.

4.If the tissue sample itself is very small, it can be sheared appropriately and directly added to the lysis buffer for cracking, and subjected to strongVorte×Fully lyse the sample. Then centrifuge and collect the supernatant for subsequent experiments. The advantage of direct lysis is that it is more convenient and does not require the use of a homogenizer, but the disadvantage is that it is not as thoroughly lysed as using a homogenizer.

5.Turbidity may occur at low temperatures, but37℃Water bath promotes its dissolution without affecting the effectiveness of use; The dissolution time should not be too long to prevent the effective ingredients from becoming ineffective.

6.A small transparent gel like substance often appears in the cracking products, which is a normal phenomenon. The transparent gel like substance contains the genomeDNAWaiting for the composite. Without testing and genomic analysisDNAIn the case of tightly bound proteins, the supernatant can be directly centrifuged for subsequent experiments; If it is necessary to detect proteins that bind closely to the genome, the transparent gel can be broken and dispersed by ultrasound treatment, and then centrifuged to obtain the supernatant for subsequent experiments.

7.The steps for cell lysis should be placed on ice or4℃conduct.

scope of application

Suitable for general biological and medical research

Product ordering information:

R301899-100ml RIPACracking solution (strong) 100ml