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    Home > Biochemistry News > Plant Extracts News > Determination of soluble protein content in plants

    Determination of soluble protein content in plants

    • Last Update: 2021-01-06
    • Source: Internet
    • Author: User
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    , principle,
    The LoWry method is a combination and development of Biuret and Folin-phenol. The principle is that after the
    protein
    solution is treated with alkaline copper solution, alkaline copper
    reager
    reacts with the peptide bonds in the protein to produce a double-shrink reaction, forming a copper-protein bond salt. After adding the phenolic reagent, under alkaline conditions, the phenolic group on the acting protein is extremely unstable, and it is easy to restore the phosphorus tungsten acid and phosphorus tantalum acid in the phenolic reagent, so that it produces a mixture of phosphorus tungsten blue and phosphorus tantalum blue. The blue depth of this solution is positively related to the protein content, so it can be used for protein content determination.
    LoWry method, in addition to making tyrosine, tryptophan and cysteine in the peptide chain, also makes the coloring effect of peptide bonds in the di-shrink method more intense, its color-showing effect is 3-15 times stronger than the use of phenolic reagents alone, about 100 times the double-shrink method. Due to the enhanced color-showing effect of peptide bonds, the deviation caused by different protein types is reduced. The LoWry method is suitable for the determination of trace proteins, and it is convenient for multiple samples to be measured at the same time. However, insoluble proteins and membrane binding proteins must be pre-treated (e.g. by adding a small amount
    SDS
    ).
    1. The principle of double lysergent (NH2-CO-NH-CO-NH2) can produce fuchsia-red enseples with copper ions in alkaline solutions, a reaction known as double-shrink reaction. Because there are multiple peptide bonds in the protein, can also react with copper ions, and the relationship between color depth and protein content in a certain range in line with Bill's Law, and protein
    amino acids
    composition and molecular weight, so the protein content can be determined by double lysine method.
    reaction mainly involves peptide bonds and is therefore less affected by protein specificity. And the use of reagents cheap and easy to get, easy to operate, can be measured range of 1-10 mg protein, suitable for the accuracy requirements of not too high protein content determination, protein content can be measured at about 0.5 mg or more. The disadvantage of the double-shrink method is the poor sensitivity and the large sample size required. Substances that interfere with this assay include buffers that are essentially amino acids or peptides, such as Tris buffers, because they produce a positive coloring reaction, copper ions are easily reduced, and sometimes red precipitation occurs.
    2. Principles of the forling-phenol method
    This method is the development of the double-shrink method, including a two-step reaction:
    (1) Under alkaline conditions, protein and copper act to produce a protein-copper compound.
    (2) This compound restores the reagent phosphoric tantalum- phosphorus tungsten acid (Folin reagent), the mixture dark blue (phosphorus tantalum blue and phosphorus tungsten blue mixture), the color depth is directly related to the protein content. This method is simple to operate, with a sensitivity 100 times higher than the double-shrink method and a quantitative range of 5-100 μg of protein. Folin reagent coloring reactions are caused by tyrosine, tryptophan, cysteine, so if the sample contains phenols, citric acid and -based compounds, there are interference. The disadvantage of this method is that there is a protein-specific effect, that is, different proteins due to the different content of lysine, tryptophan and make the color intensity slightly different, the standard curve is not a strict linear form.
    II, instruments and appliances
    test tubes; scale pipe 0.5ml 1, 1 ml 1st, 10ml 1st; quantitative sampler; round bottom burner; condensate tube 1 set (with rubber tube); micro-titration tube; small burner; microsager 50 sl 1 x light meter; 721 light meter;
    thermostat
    water bather; research, glass rod;
    centrifuge
    ; centrifuge tube.
    , reagents,
    Na2WO4.2H2O; Na2MoO4.2H2O; 85% H3PO4; Thick HCl; Li2SO4 H2O; Bromine water; phenolic indicator; Na2CO3; NaOH; CuSO4.5H2O; Potassium saloonate
    serum
    serum.
    fourth, method
    1. Preparation of
    (1) alkaline copper reagents (equivalent to double-shrink reagents
    First preparation of A solution: 4% sodium carbonate (Na2CO3) solution mixed with 0.2mol/L sodium hydroxide (NaOH) solution, etc. (2% Na 2CO3,0.1mol NaOH); B solution: 1% copper sulfate (CuSO4.5H2O) solution mixed with 2% potassium saltate solution (0.5% CuSO4.5H2O, 0.1% potassium saltate sodium). Mix the A liquid with the B liquid in a ratio of 50:1 before use. This is FolIn-phenol reagent A liquid, which can only be used for 1 day.
    phenol reagents (equivalent to Folin reagents): 100g of sodium tungstenate (Na2WO4.2H2O), sodium tantalum (Na2MoO4.2H2O) 25g, and 700ml of distilled water dissolved in a round-bottomed bottle of 1500Ml. Then add 85% H3PO450ml, thick HCl 100ml, install the reflow device (using a grinding joint, if using a cork or rubber plug, you must wrap it in tin platinum paper), so that it slowly boils 10H. After cooling, lithium sulfate (Li2SO4) is added. H2O) 150g, water 50ml, bromine water 2 to 3 drops, without the reflow device opening boiling 15min, in order to release excess bromine. Dilute to 1000ml after cooling and
    filter
    into a brown bottle and store in a refrigerator (the cooled solution is yellow and, if still green, add a few more drops of liquid bromine and continue to boil for 15min). When using the standard NaOH solution titration, with phenolic as an indicator, the titration end point from blue to gray, titration to calculate the concentration of acid. When used, add about 1x water, so that the final concentration is equivalent to 1mol/L H-plus acid, which is Folin-phenol reagent B solution (Folin reagent).
    In the determination of attention, because the phenolic reagent is only stable under acidic conditions, but the reaction of this experiment only occurs in the case of pH of 10, so when the phenolic reagent, must be mixed immediately, so that the phosphoric tantalum acid - phosphorus tungsten acid reagent can be destroyed before the reduction reaction, otherwise it will reduce the degree of color.
    (2) standard protein solution is called 25 mg of bovine serum albumin, dissolved in 100 ml distilled water, so that the final concentration of 250 mg/ml.
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