Everything you want to know about protein extraction is here!
1.What is protein extraction?
The extraction of protein refers to the process in which the treated or broken cells are put in certain conditions and solution so that the extract can be fully released.
2.Why do protein extraction?
Protein extraction is compatible with different downstream experiments, including reporter gene detection, protein purification, immunoblotting, immunoprecipitation, EMSA (also known as gel shift), footprinting, enzyme activity analysis；
a.ELISA: The soluble antigen or antibody is bound to solid phase carriers such as polystyrene, and the specific binding of antigen and antibody is used for qualitative and quantitative detection of immune reaction;
b.WB: The protein samples were separated according to molecular weight by polyacrylamide electrophoresis, then transferred to the hybrid membrane (blot), and then the target protein was detected by the first / second antibody complex;
c.IP/Co-IP: The reaction between the antigen and the antibody in the natural conformational state, and the quality of the sample treatment determines the quality and concentration of the antigen in the antigen-antibody reaction and whether the antigen is in the natural conformational state;
d.Reporter gene detection: As a reporter gene, there must be the following conditions in genetic selection and screening: (1) it has been cloned and sequenced; (2) the expressed product does not exist in the recipient cell, that is, there is no background, there is no similar endogenous expression product in the transfected cells; (3) the expressed product can be quantitatively determined.
3.What are the methods of protein extraction?
a.Solution method: such as RIPA lysate;
b.Extraction method: such as organic solvent extraction TCA precipitation;
c.Column method: centrifugal column separation.
4.Analysis and solution of Common problems in protein extraction
a.The concentration of extracted protein is low.
- Incomplete cell lysis: adding extracted Buffer or homogenate to increase incubation time;
- Incomplete protein separation: increase incubation and vortex time after adding protein separation solution;
- Insufficient cell / tissue sample size: increase the number of cells or initial tissue (mg).
b.When making the western of tissue sample protein, is there anything special to pay attention to when extracting protein?
Grinding and homogenization must be carried out, the protein solubility will be better, the centrifugation should be sufficient, and the protease activity in the tissue is stronger, and attention should be paid to inhibiting the protease activity (adding PMSF and protease inhibitor cocktail).
c.What is the method of grinding hard tissue samples?
For hard tissues of 100 mg (e.g. stomach tissue, skeletal muscle tissue), digest 5-10 min with 0.25 mg/mL trypsin on ice (incubation time varies from tissue to tissue), wash the sample with 1 mL cold lysate, remove the supernatant, add fresh and cold 1 mL lysate, transfer the sample to a precooled Dounce homogenizer and homogenize the sample on ice (usually 20-30 times).
5.The difference between RIPA method and columnar method
|RIPA method||columnar method|
|protein loss||No protein loss|
|protein denaturation||Moderation and immutability can be chosen|
|Slow||Simple and quick, 1-7 minutes to complete.|
6.Products strongly recommended by Abbkine
ExKine™ Pro Total Protein Extraction Kit for Animal Cultured Cells/Tissues（KTP3007）
Recommended index: ✮✮✮✮✮
- No protein loss;
- The operation is simple and fast, and can be completed with only 1-7min.
- The extracted protein is suitable for a variety of downstream experiments, such as Western Blot, IP, Co-IP and so on.
ExKine™ Pro Total Protein Extraction Kit for Plant Tissues（KTP3008）
Recommended index: ✮✮✮✮✮
- Convenient and fast: a "column" to the end, the whole process is 8 minutes;
- Multi-purpose: suitable for complete samples, plant tissue leaves, seeds, soft stems, roots;
- High protein yield: the total protein is extracted from 50-200 mg plant tissue, and the yield is 2-8 mg/mL;
- Easy to operate: no need for liquid nitrogen grinding, the optimized cracking buffer is more efficient to extract protein.
This promotion plan has set up a full series of protein extraction kits and organelle extraction kits with excellent quality and a huge discount, among which the column extraction kit will be given away free of charge.
Promotion time: 2022/2/9-2022/3/31
Promotion products: Protein extraction;Organelle extraction
- (1) Buy any small-size product in the list, and you can get small-size product of the a column method protein extraction kit (choose one of KTP3007/KTP3008), as well as high-value colorful sticky notes.
- (2) Buy any large-size product in the list, you can get the large-size product of the column method protein extraction kit (choose one of KTP3007/KTP3008), as well as the cute panda wrist mouse pad.
|Catalog No||Product Name||Size||Price/$|
|KTP3001||ExKine™ Nuclear and Cytoplasmic Protein Extraction Kit||50T/200T||139/299|
|KTP3002||ExKine™ Nuclear Protein Extraction Kit||50T/200T||109/249|
|KTP3003||ExKine™ Cytoplasmic Protein Extraction Kit||50T/200T||109/249|
|KTP3004||ExKine™ Total Membrane Protein Extraction Kit||50T/200T||109/249|
|KTP3005||ExKine™ Membrane and Cytoplasmic Protein Extraction kit||50T/200T||119/279|
|KTP3006||ExKine™ Total Protein Extraction Kit||50T/200T||109/249|
|BMP1001||Protease Inhibitor Cocktail (100X)||1 mL/1 mL×5||59/149|
|KTP3007||ExKine™ Pro Total Protein Extraction Kit for Animal Cultured Cells/Tissues||5T/50T||39/179|
|KTP3008||ExKine™ Pro Total Protein Extraction Kit for Plant Tissues||5T/50T||39/219|
|KTP4001||ExKine™ Nuclei Extraction Kit||50T/200T||49/149|
|KTP4002||ExKine™ Nuclei Extraction Kit (High Purity)||20T/100T||49/129|
|KTP4003||ExKine™ Mitochondrion Extraction Kit (Cultured Cells)||50T/200T||49/149|
|KTP4004||ExKine™ Mitochondrion Extraction Kit (Tissue)||50T/200T||49/149|
Cell and protein research tools
Abbkine focuses on the fields of proteinology and cytology, and is committed to the innovation and research and development of various antibodies, proteins, analytical reagents and kits, in order to become a key promoter in the development of life science research, drug development and other fields. We provide you with the favorite products of protein and immune research users, from basic immunological products, such as protein extraction and quantification, to internal reference label antibodies, primary antibodies and secondary antibodies for immunological experiments; the favorite products of cell research users, from Dyes and kits for detecting cell status, organelle extraction kits, cell substructure staining and tracking and cell metabolism detection products, to cytokine and protein detection kits for cell culture, just to help your research career !