The role of commonly used biochemical reagents in biological experiments

1. Proteinase K:

Can hydrolyze and digest proteins, especially histones that bind to DNA. Stable again in urea and SDS. The general working concentration is 50-100μg / ml, the recommended reaction buffer: 50mM Tris-HCl (pH7.5), 10mM CaCl2.

2.SDS:

Sodium dodecyl sulfate. Dissolves lipids and proteins on the cell membrane, thus dissolving the membrane protein and destroying the cell membrane, and dissociating the nuclear protein in the cell. SDS can also bind to the protein and precipitate

3.IPTG

Isopropyl-β-D-thiogalactopyranoside, commonly used for screening blue and white spots and IPTG-induced protein expression in bacteria. IPTG and lactose have similar structures, so it is similar to lactose and can be suppressed by the lactose operon Protein binding causes the spatial conformation of the repressor protein to change, the tetramer depolymerizes into monomers, and loses the ability to specifically bind to the operon, thereby releasing the repressor protein and allowing its subsequent genes to be transcribed into enzymes that utilize lactose. . Unlike lactose, IPTG is not hydrolyzed by β-galactosidase. It is often used together with X-GAL to screen blue and white spots. Very powerful inducer, very stable without being metabolized by bacteria

4.X-gal

5-Bromo-4-chloro-3-indole-β-D-galactoside is a color reagent for IPTG, which shows blue under the catalysis of IPTG. Often used with IPTG and blue and white spot screening.

5.G418:

An antibiotic, which is toxic to almost all cells, is the most commonly used reagent for stable transfection. When the neo gene is integrated into the appropriate place in the genome of the eukaryotic cell, it can start the transcription of the sequence encoded by the neo gene into mRNA, thereby obtaining the efficient expression of the resistance product aminoglycoside phosphotransferase, so that the cell gains resistance and can contain G418 is grown in selective medium. This selection feature of G418 has been widely used in gene transfer, gene knockout, resistance screening and transgenic animals.

6.DMSO

Dimethyl sulfoxide. The laboratory is often used as a liquid chromatography solvent, and as a reference when the test substance has an ultraviolet extinction value. It is soluble in all alkanes and olefins.

7. Triton X-100

TritonX-100. One of the components of the cell lysate used to protect protein activity. The ability is between NP40 and SDS, biased to NP40

8.NP-40

Very mild detergent, 1% concentration can basically destroy the cell membrane, and the effect on the nuclear membrane is weak, combined with a specific buffer can obtain cytoplasmic protein.

9. BSA

Bovine serum albumin. Mainly plays the role of maintaining osmotic pressure, PH buffering, carrier and nutrition. In serum-free culture of animal cells, the addition of albumin can play a physiological and mechanical protection role and carrier. Commonly used internal reference and antibody dilution in protein quantification.

10. Formamide

Formamide is used as a stabilizer for RNA in gel electrophoresis, and is also used to stabilize denatured single-stranded DNA in capillary electrophoresis.

11.TEMED

Tetramethylethylenediamine. Coagulant. Under neutral and alkaline pH conditions, adding TEMED can accelerate gel polymerization

12. Mercaptoethanol

A strong reducing agent. It can reduce protein disulfide bonds, so that the protein remains in a dissolved state, which is conducive to binding with SDS. The protein is dissociated into a single subunit.

13. Methylene bis acrylamide

N, N-Methylene bis acrylamide, alias MBA, also known as methylene bis acrylamide, methine bis acrylamide, N, N-methylene bis acrylamide Used as a crosslinking agent to polymerize with acrylamide to prepare polyacrylamide gel.

14. Ammonium persulfate

The role of ammonium persulfate (APS) is to provide free radicals, and then polymerize acrylamide and methylene acrylamide under the action of the accelerator TEMED.

15.B27

An additive used for the cultivation of neural liver cells and is believed to have the effect of inhibiting the growth of glial cells.

16.DTT

Dithiothreitol, a commonly used reducing agent, has both antioxidant effects. It can protect the reducing group on the enzyme molecule, maintain the reducing environment, and stabilize the enzyme activity. It is another protein denaturant that breaks disulfide bonds at different concentrations and plays different roles.

17. Freund's incomplete adjuvant and Freund's complete adjuvant

Freund's complete adjuvant is a water-in-oil emulsion containing the cell wall components of Mycobacterium tuberculosis. The adjuvant activity comes from the sustained release of the immunogen in the oil droplets and stimulates the local immune response. Freund's incomplete adjuvant is used for the initial immune stimulation. In order to reduce side effects, Freund's incomplete adjuvant without M. tuberculosis ingredients is used to enhance immunity

Complete adjuvant is a water-in-oil emulsion that can very effectively induce the production of high-titer antibodies. Freund's complete adjuvant contains the cell wall component of Mycobacterium tuberculosis, which can enhance the antibody response to the antigen. The adjuvant activity comes from the sustained release of the immunogen in the oil droplets and stimulates the local immune response.

18. Tween 20

Tween-20, commonly used non-ionic detergent.

19. Tween 80

Tweenum 80, in addition to being an emulsifier, is also used as a solubilizer for some poorly soluble drugs in water.

20. Sodium dodecyl sarcosinate decellularization reagent to prepare decellularized tissue engineering scaffold materials

21.PVPP

PVPP has a disintegrating effect on tablets, can be compatible with various water-insoluble drugs, can stabilize various suspending agents, and has the ability to form complexes and adsorption, etc., so it can be used as a pharmaceutical auxiliary. It is also used to remove polyphenols from beer.

22.PIPES

, The laboratory is used as a buffer. The full name is piperazine-N, N-bis (2-ethanesulfonic acid), and the pKa is 6.80. Does not participate in or interfere with biochemical reactions, the price is relatively expensive. Commonly used in immune and cell experiments, PIPES is sometimes used to prepare E. coli competent.

23.CTAB

Cetyltriethylammonium bromide, a detergent, can dissolve cell membranes, it can form complexes with nucleic acids, and is soluble in high salt solutions ((0.7 mol / L NaCl) when lowered When the salt concentration reaches a certain level (0.3 mol / L NaCl), it precipitates from the solution, and the CTAB-nucleic acid complex is separated from the protein and polysaccharides by centrifugation. Finally, the DNA is precipitated by ethanol or isopropanol, and CTAB is dissolved in ethanol or isopropanol and removed. CTAB method is the most commonly used method for nucleic acid extraction in laboratories.

twenty four. HEPES

Hydroxyethylpiperazine ethanesulfonic acid, a weak acid buffer, the main role is to prevent rapid changes in the pH of the medium. Under open culture conditions, the medium is free of 5% CO2 environment, CO2 gas quickly escapes, and the pH rises rapidly. If HEPES is added, the pH can be maintained at around 7.0. Generally, HEPES should be added during cloning culture

25. MOPS

A buffer solution that inhibits RNase, generally used with formaldehyde (for deforming RNA) for RNA denaturation electrophoresis

26. Guanidine hydrochloride

Guanidine hydrochloride can break the hydrogen bonds in the protein structure, which increases the solubility of non-polar molecules, including amino acid side chains, and reduces hydrophobic interactions.

27. PMSF

Phenylmethanesulfonyl fluoride, Chinese name is benzylsulfonyl fluoride. Used to inhibit protease. PMSF seriously damages respiratory mucosa, eyes and skin, and is fatal if inhaled, swallowed or absorbed through the skin. Once the eyes or skin come into contact with PMSF, it should be rinsed with plenty of water immediately. Any clothing contaminated with PMSF should be discarded. PMSF is unstable in aqueous solution. It should be added to the lysis buffer from the stock solution before use. The rate of PMSF activity loss in aqueous solution accelerates with the increase of pH, and the deactivation rate of 25 ℃ is higher than 4 ℃. At a pH value of 8.0, the half-life of a 20 μmmol / l PMSF aqueous solution is approximately 85 min, which indicates that the PMSF solution is adjusted to alkaline (pH> 8.6) and left at room temperature for several hours before it can be safely discarded.

28. BPB

pH indicator, yellow at pH = 3.0, blue-violet at pH = 4.6. Acid-base indicator; non-aqueous indicator, protein electrophoresis staining; virus test, etc.

29. Trypan blue

TrypanBlue, or trypan blue, is a cell-active dye commonly used to detect the integrity of cell membranes

30. Xylene Green FF

Tracer dye for agarose polyacrylamide gel electrophoresis, easily soluble in water and alcohol, and loading buffer can be configured with bromfen blue in a certain ratio

31. Acridine orange

It is a fluorescent pigment. There is a difference in the amount of DNA and RNA binding in the cell. It can emit different colors of fluorescence. The amount of DNA binding is less green and the amount of RNA binding is more orange or red. The dye has membrane permeability and can penetrate cell membranes to stain nuclear DNA and RNA. Observed under a fluorescent microscope, acridine orange can penetrate the normal cell membrane to make the nucleus show green or yellow-green uniform fluorescence; while in apoptotic cells, due to chromatin condensation or breakage into fragments of different sizes, the formation of small apoptosis body. Acridine orange makes it densely dyed with yellow-green fluorescence, or yellow-green debris particles; and the yellow fluorescence of necrotic cells weakens or even disappears. Acridine orange AO is often used in combination with ethidium bromide EB, because EB only stains dead cells to produce orange fluorescence, which can distinguish normal cells, apoptotic cells and necrotic cells. It can also be used as frame shift Mutant mutagens can be embedded between two adjacent base pairs, so that in the process of DNA replication, the DNA strand will be increased or deleted by one base, causing frameshift mutations. Acridine orange stain is toxic, wear gloves and avoid light during operation.

32. EDTA and EGTA

EDTA--EDTA is a representative substance of chelating agent. It can form stable water-soluble complexes with alkali metals, rare earth elements and transition metals. In addition, EDTA can also be used to detoxify harmful radioactive metals from the body quickly. It is also a water treatment agent. EGTA-ethylene glycol bis (2-aminoethyl) tetraacetic acid can be directly titrated with EGTA when measuring Ca2 + in the presence of .Mg2 +, the titration error is less than 0.3%, which improves the selectivity than EDTA titration. In addition, it is more selective The stability of complexes formed by ions is generally worse than that of corresponding EDTA complexes.

33.TRICINE

N- [tris (hydroxymethyl) methyl] glycine, the common electrophoresis system is the Tris-glycine system; Tris-Tricine electrophoresis is used for the electrophoresis of peptides and proteins with a molecular weight of less than 10kDa, which can obtain better separation results. Use the former instead.

34. PVP40

CO-N in PVP40 = has a strong ability to bind phenolic compounds, and it forms a stable complex with it. It is often used to remove phenols when extracting RNA.

35. Sodium deoxycholate

Ionic detergent, the role is: 1,

Lyse cells; 2.

Dissolve proteins, especially those that are difficult to dissolve in water, such as membrane proteins; 3. It is very suitable for WB, but it needs to be used with caution in Co-IP

36, 6-BA

Used in cell culture, cytokinin.

37. Iodoacetamide

Also called iodoacetamide, 2-iodoacetamide. Used in organic synthesis, alkylating reagents for histidine and cysteine ​​in proteomics, for peptide sequencing and enzyme inhibitors, etc. Grade reagent

38. Naphthaleneacetic acid

NAA, a broad-spectrum plant growth regulator, can promote cell division, induce the formation of adventitious roots, change the proportion of male and female flowers, increase the fruit setting rate, etc.

39, Safranin O

Saffron T;

Redox indicator; biological staining; acid-base indicator; titration analysis of nitrite.

40 、 TTC

2,3,5-triphenyltetrazolium chloride (red tetrazolium), mostly used for drug analysis and agarose additives, add an appropriate amount of TTC (0.5% TTC 1ML to 100ML agar) in the counting agar, Bacterial colonies grow into a red color, which is very meaningful for removing the interference of particulate matter in the food background.

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