Equipment preparation

centrifuge; Oscillator; Vortex mixer; Pipetting; The refrigerator; Ice boxes

Reagent preparation: acetonitrile; Protein quantification kit

Material preparation

centrifugal tube; Suction head; disposable gloves

Method of use:

1. Other protease inhibitors can be added according to your own experimental needs.
2. The centrifuge speed has two ways of relative centrifugal force (RCF, ×g) and speed per minute (RPM). Some centrifuges have RPM and ×g display switching, but some centrifuges do not have automatic switching function. The following formula needs to be used for conversion: g=r×1.118×10-5×rpm2 (r is the effective centrifugal radius, that is, the length from the centrifuge axis to the center of the bottom of the centrifuge collection tube in centimeters)

For example, if the rotational speed is 3000rpm and the effective centrifugal radius is 10 cm, the relative centrifugal force (RCF, ×g) is =10×1.118×10-5×30002=1006.2 (×g).

Operation:

1, 200ul of the serum sample to be processed was centrifuged at 15000×g for 15 min at 4 ° C.

2, carefully remove the upper lipid layer, draw the middle tier serum 50 ul.

3, add in 150 ul reagent A, thoroughly incorporated.

1. Let stand at -20℃ for 30-90 min.

5, Centrifugation at 15000×g for 20 min at 4 ° C.

1. Carefully suck out the supernatant. The supernatant was discarded and the precipitate was retained.
2. 500ul of reagent B in an ice bath was added to the precipitate and mixed with gentle shaking. 15 min is placed on the ice.

8, Centrifugation at 15000×g for 20 min at 4 ° C. Carefully remove the supernatant, discard the supernatant, and retain the precipitate.

1. 500ul of reagent B in an ice bath was added to the precipitate and mixed with gentle shaking. Leave on ice for 15min.

10, Centrifugation at 15000×g for 20 min at 4 ° C. Carefully remove the supernatant, abandon supernatant, reservation precipitation.

11, precipitation will retain the centrifugal tube open let stand for 15 minutes. To dry.

12, add 100-400ul reagent C to the precipitate and mix thoroughly. Oscillation for 5-10 minutes.

13, centrifuged at 12000×g for 10 min at 4 ° C. The precipitate was discarded and the supernatant was removed.

14, in addition to the supernatant is the albumin serum protein samples.

Albumin /IgG removal kit provides a full set of reagents suitable for rapid removal of albumin from serum/plasma samples.

Serum and plasma proteins are the focus of human index detection, and also the focus of scientific research. Commonly used techniques such as electrophoresis, 2D electrophoresis, liquid chromatography and mass spectrometry have been widely used in these studies. These techniques have limitations on the total amount of protein in the sample, and more than 50% of serum proteins are albumin, which will cause great interference to the detection of other low abundance proteins, which forces us to find ways to enrich the target protein and remove albumin.

The protein extracted by this kit can be used for downstream protein research experiments such as Western Blotting, protein electrophoresis, immunoprecipitation and ELISA.

The protein samples extracted by this kit contain a high concentration of salt components and cannot be directly used for 2D electrophoresis.

The protein extracted by this kit has an effect on the activity of the protein, and can not be used for downstream protein research experiments such as enzyme activity determination. 1. Simple, no special equipment other than centrifuge is required, and multiple samples can be processed in parallel.

2, high efficiency, one can effectively remove about 60% of the albumin, conducive to the detection of low abundance protein.