Drug student experiment: hydrophobic layering

the principles and methods of hydrophobic layering through experiments.
: hydrophobic
(Hydrophobic Interaction Chromatography, HIC) is a more commonly used method for separating biological large molecules such as
proteins
and
peptides
based on differences in hydrophobicity on molecular surfaces.
Proteins and peptides and other biomolegiles are often exposed to hydrophobic groups, which we call hydrophobic patches, which can be combined with hydrophobic interactions with hydrophobic laminate media.
Different molecules because of different hydrophobic, they and hydrophobic layering medium between the hydrophobic force is different, hydrophobic layering is based on this principle to separate purified proteins and peptides and other biological molecules.
ion strength in the solution can enhance the hydrophobic action between biomolecules such as proteins and peptides and hydrophobic layering media. Using this property, the sample to be separated is adsorbed to the hydrophobic layering medium at high ion strength, and then the sample is desorpted linearly or in stages to reduce the ion strength selectively. Substances with weak hydrophobicity are puried in solutions with higher ion strength, and when ion strength decreases, hydrophobic substances are subsequently puried.
Phenyl-SepharoseTM6 Fast Flow is a hydrophobic layering medium that is supported by cross-linking
agar
sugars, which are combined with benzene-based co-prices. As a hydrophobic complex, benzene can be hydrophobic with hydrophobic substances.The Experimental Materials
1.Experimental equipment
a layering column (1.6X20 cm); constant current pump; gradient mixer;
test tube
and test tube holder; ultraviolet
dicing photomedia
2.Experiment
Reagent
(1) Hydrophobic Layering Media: Phenyl-SepharoseTM6 Fast Flow (2) Solution A:0.1M Na2HPO4, pH7.0 (3) Solution B:0.1M Na2HPO4, pH7.0, 1.7M (NH4) 2SO4 (4) Protein sample dissolved in Solution B
Experimental Operations
1.Layering media preparation: Phenyl-SepharoseTM6 Fast Flow hydrophobic analysis medium is stored in 20% ethanol, the layering medium is removed, and the ethanol solution is poured out. Add solution A, the volume of the solution is about 1/4 of the total volume.2. Mounting column: wash the column, fixed to the iron frame table, the bottom of the column with a spiral clip clamping. Add solution B, open the lower mouth to allow the solution to flow out, drain the residual bubbles, the column retains a height of about 2 cm solution. Gently stir the prepared layering medium, drain with a glass rod, and slowly add the layering medium to the column along the inner wall of the column.
until the layering medium is deposited in the column at a height of more than 1 cm, open the lower port. When the height of the column bed reaches 6-8 cm, close the lower port, the column is installed as once possible, to avoid the interface.3. Column balance: Balance the volume of 1-2 beds with solution B. Be careful to always keep the layering medium in the solution and do not dry the column.4. Sample: Take samples to add a well-balanced analysis column, and collect the dithography column lower mouth outflow parts, adjust the flow rate of 1 ml/min, 3 ml per tube.5. Washing: Washing 1 bed volume with solution B, washing off the sample without adsorption. Collect the components that flow out of the lower mouth of the column.6. Wash and collect: Gradient mixer with 250 ml solution A on the left and 250 ml solution B on the right. Collect the eals according to 100%-0%1.7M (NH4)2SO4
gradient to dissodiate 500 ml.7. Detection: Take samples from each collection tube and measure UV absorption at 280 nm.8. Hydrophobic analysis medium cleaning and preservation: the layering medium is first washed with water, then washed with 0.5 MNaOH, and finally washed to neutral with water. The treated layering medium is placed in 20% ethanol and stored at 4 degrees C.
results
the horizontal coordinates of each collection tube, and the UV absorption value at 280 nm is the ordinate, and the excruced curve is obtained. Analyze the results of the experiment and discuss them.
is the
between hydrophobic and inverse stratography?