Recombinant protein of human high temperature demand factor A1 (HTRA1)

Human high temperature demand factorA1 (HTRA1) recombinant protein

Product Name: High Temperature Demand FactorA1 (HTRA1) recombinant protein

English nameRecombinant High Temperature Requirement Factor A1 (HTRA1)

HtrA; L56; ORF480; PRSS11; HtrA Serine Peptidase 1; Protease,Serine,11; IGFBP5-Protease; High-temperature requirement A serine peptidase 1

Model:CS-D003

Enzymes and kinases

Species:Homo sapiens (Human)

Source: Prokaryotic expression

hostE.coli

Endotoxin level:<1.0 EU/μ g (measured by LAL method)

Subcellular localization: secretion, cytoplasm

Predicted molecular weight:47.2kDa

Actual molecular weight:47kDa (please refer to the instruction manual for differential analysis)

Fragments and tags:Gly204~Leu364 with N-terminal His and GST Tag

Composition of buffer solution:20mM Tris, 150mM NaCl buffer (pH 8.0, containing 1mM EDTA, 1mM DTT, 0.01% SKL, 5% Trehalose, and Proclin300)

Characteristic: Freeze dried powder

Purity:> 90%

Isoelectric point:6.6

Application:Positive Control; Immunogen; SDS-PAGE; WB.

Specifications:10μg50μg200μg1mg5mg

1. Tissue protein extraction

1) Required equipment: ice maker, marker pen, two sets of 1.5ml EP tubes (high temperature and high pressure treatment), a large ice box, a 1.5ml EP tube box, gloves, ophthalmic scissors (high temperature and high pressure treatment), fresh tissue or tissue stored in a refrigerator at -80 ℃, PBS (high temperature and high pressure treatment) stored in a refrigerator at 4 ℃, fluid transfer gun, suction head (high temperature and high pressure treatment), two sets of grinding rods (high temperature and high pressure treatment), palm centrifuge, filter paper, triple decontamination lysate, fluorine benzylsulfonate (PMSF, a protease inhibitor, highly toxic), centrifuge, beaker, 2 × SDS gel sample loading buffer, disulfide threitol (DTT), oscillator, foam board.

a. Ice making machine for making ice;

b. Mark the two sets of EP tubes with a marker pen;

c. Ice the large ice box and EP tube box, place one set of marked EP tubes in the large ice box, and place the other set of marked EP tubes in the EP tube box. Wear gloves, put on the EP tube box, and use ophthalmic scissors to cut fresh tissue the size of soybeans (about 100mg) or store it in a -80 ℃ freezer;

d. Take out PBS stored in a refrigerator at 4 ℃, add 1ml PBS dropwise to the EP tube, cut and shred the ophthalmic tissue (fast action), centrifuge on the palm, discard the supernatant, and then add 1ml PBS dropwise to the EP tube. Grind with a grinding rod, centrifuge at 3000 rpm for 10 minutes, discard the supernatant, filter paper as much as possible to absorb the residual liquid at the tube mouth, estimate the volume of sediment in the EP tube, and perform the above steps on ice as much as possible;

e. Take out the three decontamination cracking solution from the 4 ℃ refrigerator, take out PMSF from the -20 ℃ refrigerator and place it in a large ice box. Drop 3-5 times the volume of the sediment into the EP tube (usually 5 times), then add PMSF (the ratio of the two is 94:6), grind with a grinding rod (fully crack on ice for 20-30 minutes), and all the above steps need to be carried out;

f. Centrifuge pre cooling, centrifuge the fully decomposed tissue solution at 4 ℃ and 10000 rpm for 10 minutes;

g. Wash the beaker with tap water, pour in distilled water, and boil it on an electric stove;

h. Prepare 2 × SDS gel sampling buffer solution (stored at room temperature), take out DTT stored in the refrigerator at -20 ℃, place it in a large ice box, use a pipette gun to quantitatively suck the centrifuged supernatant into another set of EP tubes (do not suck the sediment in the lower layer), add 2 × SDS and DTT according to the supernatant: 2 × SDS: DTT=1:0.8:0.2, centrifugate on the palm, then carefully insert the EP tube into the foam plate, put it into the boiling beaker and boil for 6-8 minutes (the power of the electric furnace should not be too high to prevent the tube cover from exploding);

i. Take out the foam board with tweezers, put it in a large ice box to cool for 10 minutes, centrifugate it on the palm, and then put it into a -20 ℃ refrigerator for standby.

Attention: Single detergent lysis buffer or cell lysis buffer can also be used as appropriate.

2. Protein extraction from adherent cells (cell protein content is generally about 1x10-9mg/cell)

1) Required equipment: ice maker, cell scraper, marker pen, two sets of 1.5ml EP tubes (high-temperature and high-pressure treatment), two large ice boxes, gloves, culture bottles full of cells, PBS stored in a refrigerator at 4 ℃, triple decontamination lysate, benzyl fluoride sulfonate (PMSF, a protease inhibitor, highly toxic), pipette gun, suction head (high-temperature and high-pressure treatment), filter paper, centrifuge, beaker, 4 × SDS gel loading buffer, dithiothreitol (DTT), foam plate.