RNA Binding Protein Immunoprecipitation (RIP)Kit
RNA Binding Protein Immunoprecipitation (RIP)Kit
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| Usage | 1, sample cracking 1.1 animal cell (1) to prepare 405 ul Complete RIP Lysis Buffer (400 ul RIP Lysis Buffer + 4 ul Protease Inhibitor (100 x) + 1 ul RNase Inhibitor); (2) 2xl07 cell samples were collected and washed by adding 2 mL PBS. After centrifugation, the supernatant was discarded to collect cell precipitation, and 1/4 cell samples were reserved for RNA extraction and purification in Input group (stored in nuclease-free EP tube at -80°C). (3) 400uL Complete RIP Lysis Buffer was added to the remaining cell precipitate and resuspended, and the precipitate was blown and mixed 10 times with a pipettes gun to fully lyse the cells. The cells were bathed in ice for 30min, vortexed every 5min, 10s/ time. 20% power, ultrasonic 3 s, intermittent 3 s. After centrifugation at 12000rpm for 10min at 4 °C, the supernatant was removed, recorded as Lysis, and divided into triplicate according to 150uL (IP), 150uL (lgG), and 100uL (Input), and the Input sample was stored at -80°C until use. 1.2 animal tissues (1) to prepare 405 ul Complete RIP Lysis Buffer (400 ul RIP Lysis Buffer + 4 ul Protease Inhibitor (100 x) + 1 ul RNase Inhibitor); (2) grinding: take fresh organizations or low temperature (about 0.3 g), put in precooling of mortar after sterilization, to join the liquid nitrogen quick grinding to powder, and collect about a quarter of the tissue samples reserved for subsequent Input set of RNA extraction and purification (using EP pipe storage without nuclease, - 80 ° C). (3) to join in mortar samples 250 ul Complete RIP Lysis Buffer, continue to grind on the ice samples into 5-10 min to the exquisite homogenate, transferred to the new EP in the tube, Then add the remaining 150uL Complete RIP Lysis Buffer to the mortar to collect the remaining samples, and transfer them to the EP tube as well. (4) The EP tube containing the sample homogenate was fully lysed for 30min on ice, vortexed every 5min, 10s/ time. After the lysis was completed, the cell ultrasonic breaker was used for 8-10min in an ice bath, with 20% power, ultrasonic 3s, intermittent 3s. (5) Centrifugation at 12000rpm for 10 min at 4°C, the supernatant was removed, and then the volume of RIP Lysis Buffer was added to the supernatant to 400uL, mixed, and divided into three parts according to 150uL (IP), 150uL (lgG), and 100u L (lnput). Input samples were stored at -80°C until use. 2, preparation of magnetic beads (1) Two 1.5mL EP tubes were prepared and labeled with lgG tube and IP tube, respectively. The original ProteinA/G Magnetic Beads tube was upside down 10 times. After the magnetic beads and liquid were mixed, 30uL were taken out into lgG tube and IP tube, respectively. (2) for every 300 ul tube join RIP for Wash Buffer, pipetting gun beat five blending, placed in a magnetic rack let stand for 1 min, abandon in qing dynasty, the three steps; (3) The beads were resuspended in 300 ul of RIP Wash Buffer. 3, magnetic beads in combination with antibodies (1) 3-5ug target antibody was added into IP tube, and 3-5ug target antibody of the same host lgG was added into lgG tube, and then incubated in a silent mixer for 2 hours at room temperature. (2) Put the two tubes on the magnetic rack for 1min, and discard the supernatant; (3) to join 300 ul RIP for Wash Buffer, 5 times, percussion and mixed with pipetting gun in magnetic rack let stand for 1 min, in qing dynasty, the steps for 3 times. 4, RNA binding protein immunoprecipitation (1) Prepare 1.8mL RIP Buffer (1720uL RIP Wash Buffer+70uL 0.5M EDTA+10uL RNase Inhibitor); (2) the lgG obtained from step 3 respectively added into the pipe and the IP 900 ul RIP Buffer, 150 ul Lysis obtained from step 1 on mute mixer, 4 ℃ incubation for the night; (3) put the two tubes in magnetic rack let stand for 1 min, abandon the supernatant; (4) Add 300uL RIP Wash Buffer to each tube, blow and mix 5 times with a pipetting gun, stand on a magnetic rack for 1 min, discard the supernatant, and this step is performed 5 times. (5) will join the 300 ul RIP each tube for Wash Buffer, pipetting gun beat five blending, take 100 ul mixture to the new tube, marked as (1) tube, the remaining liquid for (2) pipe, the 4 tubes placed in a magnetic rack let stand for 1 min, abandon supernatant, (1) tube plus 100 ul Elution Buffer, After boiling for 10min, the supernatant was taken to a new tube, and 10uL of 6×Loading Buffer was added to mix for WB detection. ② The tube was used to purify RNA, and the grouping Settings are shown in the table below:
(1) step 1 reserved Input samples and lgG - (2) pipe, IP - (2) each to join 500 ul Trizol, vortex blender, let stand at room temperature for 5 min, then add 100 ul chloroform, vortex blending, 4 ° C 14000 RPM centrifuge for 10 min, taking the upper water phase (about 300 ul) to the new tube, marking; (2) Add 50uL Salt Solution, mix with 550uL isopropanol, let stand at -80°C for 2-4h (or let stand overnight), place at 4°C to thaw before use; (3) 4 ° C 14000 RPM centrifuge for 10 min, abandon the supernatant; (4) 500uL of 75% ethanol was added, centrifuged at 4°C at 14000rpm for 10min, and the supernatant was discarded. This step was repeated 3 times. (5) Open the cover and dry at room temperature, add 10-20uL DEPC H2O, blow with pipetting gun to completely dissolve the RNA, and store at -80°C for later use. |
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| Synonym | RNA Binding Protein Immunoprecipitation (RIP)Kit | ||||||||||||||||||||||||||||||||||||
| Description |
1. Experimental principle RIP (RNA Binding Protein lmmunoprecipitation) is a technique for studying the binding of RNA to proteins in cells. It is a powerful tool to understand the dynamic process of post-transcriptional regulatory network. This technology mainly uses the specific antibody of the target protein to precipitate the corresponding RNA-protein complex, and then after isolation and purification, the RNA bound to the complex is verified by q-PCR or high-throughput sequencing.
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| Composition |
Note: 1, need to bring your own Trizol, 75% ethanol, chloroform, isopropyl alcohol, reverse transcription kit and fluorescent dye; 2, 6 t for a single set (1 set of IP or 1 set of IgG) immune precipitation experiment, the operation steps behind the 1 set contains the IgG and IP, it takes 2 t reagent. |
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| General Notes | The kit is mainly used in animals. | ||||||||||||||||||||||||||||||||||||
| Storage Temp. | Each component was stored according to the corresponding storage temperature, and the validity period was 1 year. |
