For a perfect westernblot experiment, the protein is separated in a polypropylene gel according to the natural molecular weight, and the protein is well transferred from the gel to the solid support and then immunodetected by specific antibodies. For transfer, the wet transfer method is better because both the gel and the film can be completely immersed in the transfer buffer. Under semi-dry conditions, the transfer occurs between the two electrode plates. This type of transfer device may affect the transfer efficiency of the protein and the retention capacity on the film. For optimizing the transfer conditions of the protein, the advantages and disadvantages of each system should be taken into account. Wet turn For a wet transfer process, create a sandwich structure in the order of sponge, filter paper, film, glue, filter paper, and a second sponge. Before assembly, all parts (including glue) need to be placed in the transfer buffer for balance, then the stacked sandwich structure is clamped on the transfer device with the transfer clamp, connected to the electrode device, and the protein is transferred to the buffer. Transfer from the gel to the membrane. Wet rotation can be used in most western blot applications. Semi-dry For semi-dry rotation, stack assembly (filter paper, glue, film needs to be pre-balanced in the transfer buffer) is placed on the bottom of the device electrode plate, and the upper plate is covered, and the high-current current passes through the sandwich structure. The transfer speed is faster, generally less than 1h. Transfer precautions 1. When preparing the transfer buffer, be sure to pay attention: the inconsistency of small components may affect the transfer 2. Use high quality, high purity methanol solution. If the quality of methanol is not good, it will affect the efficiency of film transfer. 3. Do not dilute the transfer buffer 4. Do not adjust the PH value of the transfer buffer 5. In order to get the best transfer, the buffer should use the new one. 6. The device is kept clean 7. Use a certain thickness of glue (0.5-0.75) 8. If PVDF membrane is used, the membrane needs to be soaked in 100% methanol before transfer. 9. Transfer pad, filter paper, glue and film need to be balanced in the transfer buffer for at least 15min 10. Drive away the air bubbles between the film and the glue during transfer. 11. Make sure the electrodes are connected correctly to avoid unnecessary losses 12. Use pre-dyed molecular weight marker to monitor transfer 13. For difficult-to-transfer proteins, the concentration of methanol and SDS can be adjusted. SDS can enhance the migration of proteins from the gel and inhibit the binding to the membrane. Methanol promotes protein and membrane binding but hinders protein migration from the gel.
The RNA purification kit is used to purify and recover RNA molecules transcribed in vitro and total RNA extracted from various materials, which can effectively remove contaminating impurities in RNA samples. The recovery rate of this product can reach 80%, and the OD260/OD280 ratio of the obtained RNA is generally about 2.0, which can be directly used in subsequent sensitive experiments (such as microarray analysis, fluorescence RT-PCR, etc.). With the deepening of transcriptomics, the complexity of RNA types, expression regulation and functions is far beyond our imagination. Covid-19 Nucleic Acid Extraction Reagent,Nucleic Acid Extraction Reagent Kits,Dna Purification Kit,Rna Purification Kit Jilin Sinoscience Technology Co. LTD , https://www.jilinsinoscience.com
Removing ribosomal RNAs that account for more than 80% helps to focus sequencing on less abundant but informative RNAs. At present, the removal of rRNA is mainly through the combined use of probe and RNase H. The processed RNA will be mixed with many digestion products, enzymes and ions, which is not conducive to the subsequent construction of RNA library. Take the Columnar RNA Purification Kit as an example. Trizol is a ready-to-use reagent that can be used to purify total RNA from tissues and cells. This is a single-phase solution of phenol and guanidine isothiocyanate that facilitates lysis of tissues and cells, inhibiting RNases to maintain RNA integrity.