Biochemistry and Molecular Biology Laboratory Manual

Author: Shao Xueling et al. / Country:
Publisher:
Publishing Time: 2003-01-01
Features:
2. DNSylation of N-terminal amino acids of proteins Take 0.5 mg of protein sample and place it in a stoppered glass tube. Dissolve it with a small amount of water, then add 0.5 ml of 0.2 mol/L sodium bicarbonate solution. Add 0.5 ml of DNS-CI acetone solution and adjust the pH to 9.0-9.5 with triethylamine. Stopper the tube and react at 40°C in an oven for 2 hours, or let it stand at room temperature (around 25°C) for 2-4 hours to generate DNS-protein.
3. Hydrolysis of DNS-protein After the DNSylation reaction is complete, evaporate the acetone under vacuum. Add 0.5 ml of 6 mol/L hydrochloric acid to dissolve the DNS-protein. Transfer the entire solution to a hydrolysis tube. Suck out the vacuum and seal the tube. Hydrolyze at 111°C in an oven for 18-24 hours. After opening the tube, evaporate the hydrochloric acid, add a small amount of water, and then evaporate to dryness. Repeat 2-3 times to remove all hydrochloric acid.
4. Extraction of DNS-amino acids Take the above hydrolysis product, add 0.5 ml of water, and adjust the pH to 2-3 with 1 mol/L hydrochloric acid. Add 0.5 ml of ethyl acetate for extraction. The layers can be separated in a long dropper. Repeat the extraction 2-3 times, combine the upper layer of the extract into a small test tube, remove the ethyl acetate, and place it in a desiccator for later use.
5. Chromatography and detection of DNS-amino acids The generated DNS-amino acids and standard DNS-amino acids are subjected to polyamide film chromatography separately.
(1) Preparation of polyamide film Cut the polyamide film into 7 cm × 7 cm squares. Draw two perpendicular base lines at a distance of 0.5 cm from the edges, with their intersection point as the origin. If only single-phase chromatography is performed, draw only one base line and mark a sample point every 1 cm along the line.
(2) Spotting Use a capillary to take the sample and spot it at the designated position. The spotting diameter should be less than 2 mm. If spotting is repeated multiple times, spot once and dry once.
(3) Development Roll the spotted polyamide film into a cylindrical shape. The sample is placed inside the cylinder, secured with a coil. Place it in a small chromatography tank (a small desiccator with a can be used as a substitute). Add 5-10 ml of the developing solvent I to the tank and develop until the solvent front reaches about 0.5 cm from the top (approximately 20 minutes). Remove the film and dry it. For two-dimensional chromatography, after the first development, completely dry the film (sometimes it needs to be air-dried overnight to fully dry). Turn the polyamide film 90 degrees and develop with solvent II. To distinguish DNS-threonine or to differentiate DNS-aspartic acid from DNS-glutamic acid, after the second development, dry the film and then develop with solvent III in the same direction, only up to half the height. To distinguish ε-DNS-lysine, α-DNS-histidine, and DNS-arginine, after the second development, dry the film and then develop with solvent IV in the same direction.
(4) Detection of DNS-amino acids After development, remove the film and dry it with a hairdryer. Examine it under a UV lamp at 360 nm or 280 nm. DNS-amino acids emit yellow fluorescence. Additionally, there may be other colored spots, such as DNS-OH showing green fluorescence. By comparing the chromatogram of the sample with that of standard DNS-amino acids, the types of DNS-amino acids in the sample can be identified.
[Thought]
1. What are the advantages of thin-layer chromatography compared to other chromatographic methods?
2. What is the basis for selecting the developing agent?
Experiment Nine: Isolation and Purification of Animal Genomic DNA
[Objective and Requirements] Master the basic principles and methods of preparing large amounts of animal genomic DNA using the salt precipitation method.
[Experimental Principle] The separation is based on the difference in solubility of ribonucleoproteins (RNP) and deoxyribonucleoproteins (ODNP) in a solution of sodium chloride at a certain concentration. Proteins are then removed using a protein denaturing precipitant to release the nucleic acids, which are subsequently precipitated from the solution due to the insolubility of nucleic acids in ethanol, achieving separation and purification. At 0.14 mol/L sodium chloride, RNA nucleoproteins (RNP) have high solubility, while DNA nucleoproteins (ODNP) have low solubility. Conversely, at 1 mol/L sodium chloride, ODNP has high solubility, while RNP has low solubility, thus separating DNA and RNA nucleoproteins. After nucleoprotein separation, protein denaturing precipitants (such as chloroform-isoamyl alcohol, sodium dodecyl sulfate, or hot phenol) can be used to remove proteins, releasing nucleic acids, which then precipitate from the solution. Animal liver contains ribonuclease and deoxyribonuclease, so the temperature must be kept low, and activation ions such as Mg2?, Fe2?, and CO? must be prevented.
[Experimental Apparatus and Reagents]
1. Reagents:
(1) SC buffer: Dissolve 2.94 g of sodium citrate and 9.0 g of sodium chloride in 1 L of distilled water. Adjust the pH to 7.0 with hydrochloric acid.
(2) 5% SDS solution
(3) 95% ethanol, chloroform, isoamyl alcohol
2. Apparatus: Freezing centrifuge, tissue homogenizer, beakers, graduated cylinders, glass rods, Erlenmeyer flasks, centrifuge tubes.
[Experimental Procedure]
Take 4 g of fresh pig liver, wash away the blood with SC solution, and chop it into small pieces at low temperature. Add 8 ml of the above solution and continue homogenizing. Centrifuge the homogenate at 4000 rpm for 10 minutes. The upper layer is the RNP extract, and the lower layer is the DNP and cell debris. Transfer the upper layer (or retain it for RNA preparation) and repeat the extraction twice with an additional 5 ml of SC solution to reduce the influence of RNP on DNP extraction. Transfer the lower precipitate to an Erlenmeyer flask, add 20 ml of the same solution, mix well, add 4 ml of 5% SDS, and mix well. Add 15 ml of a chloroform/isoamyl alcohol (20/1) mixture, mix while adding solid sodium chloride to achieve a final concentration of 1 mol/L, and oscillate for 30 minutes. Centrifuge at 4000 rpm for 20 minutes. Carefully observe the three layers: the upper layer is the aqueous phase (I) where DNA dissolves, the middle layer is a milky white protein precipitate, and the lower layer is the chloroform layer. Carefully aspirate the upper layer with a pipette. Repeat the protein removal process in the same manner until no protein precipitate remains in the middle layer. Measure the volume of the upper aqueous phase and add an equal volume of cold ethanol (95%) to a beaker. Stir gently (in one direction) until DNA fibers wrap around the glass rod. When all the DNA has wrapped up, squeeze it dry and wash it once more with anhydrous ethanol. Place it in a desiccator to dry and weigh to calculate the yield.