Antibody & Protein FAQs

This kit provides a full set of reagents for classic WB experiments, including the reagents required from protein extraction to testing of result. It has the advantages of simple operation, high detection sensitivity, clear background, and strong system stability.

Since PVDF membranes have two pore sizes, which are suitable for transferring target proteins of different molecular weights, we have designed two specifications in order to meet different experimental needs. E-IR-R304A (PVDF membrane 0.45 μm pore size) is recommended for the detection of proteins with a molecular weight greater than 20 kDa E-IR-R304B (PVDF membrane 0.22 μm pore size) is recommended for the detection of proteins with a molecular weight less than 20 kDa.

The E-IR-R305 gel kit is not included in E-IR-R304A and E-IR-R304B, and the gel kit needs to be purchased separately;

E-IR-R304A&E-IR-R304B kit contains filter paper, but does not contain sponge pad

The instruments required for WB experiments include ultrasonic cell crusher or a sonicator, water bath, pipette and pipette tip, centrifuge, electrophoresis tank, SDS-PAGE electrophoresis instrument, film transfer instrument, shaker, and WB imaging system instruments.

Both of these are enzyme inhibitors and their function is to prevent protein degradation. PMSF is often used as a protease inhibitor in biochemical and molecular biology experiments. It is an inhibitor of serine proteases and can inhibit the activities of trypsin, chymotrypsin and AChE. Na3VO4 is a phosphatase inhibitor, inhibiting ATPase, alkaline phosphatase and tyrosine phosphatase. The two products in the kit are 100X and should be added to the PIPA lysis buffer at a ratio of 1:100 before use.

After RIPA lysis buffer lyses the cells, the DNA in the nucleus diffuses into the sample relatively completely, forming a sticky liquid. In this case, it can be sonicated after lysis at 35~40% power using ice bath for a total of 1 min, sonicate for 2 s, and keep the interval 2 s to ensure that the cells were fully lysed and to reduce the viscosity of the sample.

Approximately 1 x 106 cells can be lysed. If a well plate is used, it is recommended to add 100-150 μL RIPA lysis buffer to each well of the 6-well plate.

No, the cell lysis buffer for co-immunoprecipitation is a lysis buffer that lyses cells under non-denaturing conditions. Conventional RIPA lysis contains SDS, which will denature the protein and affect the interaction between proteins. It is recommended to use denaturant-free lysis buffer for co-immunoprecipitation.

If you are making a phosphorylated antibody, you must add Na3VO4 inhibitor. If you are making a normal antibody, you can only add PMSF (protease inhibitor).

If the equipment does not have a medium or lower heating option, you can choose a power of about 100-200W to maintain the liquid in a simmring state (that is, just keep the liquid in a gentle bubble state).