Serum Mucin Assay

Summary

When 0.6 mol/L perchloric acid is used to determine the protein in serum, mucin is not precipitated and remains in the filtrate, then phosphotungstic acid is added to precipitate the mucin, and then the protein content is determined by phenol reagent.

Operation method

Serum Mucin Assay

Principle

When 0.6 mol/L perchloric acid was used to determine the proteins in serum, the mucin was not precipitated but remained in the filtrate, and then phosphotungstic acid was added to precipitate the mucin, and then the content of the proteins was determined by phenol reagent.

Move

I. Experimental reagents:

1. 154 mmol/L sodium chloride solution.

2. 1.8mol/L perchloric acid: take 28ml of perchloric acid with 70%-72% water content, dilute 200ml with distilled water and calibrate it.

3. 17.74mmol/L phosphotungstic acid solution, weigh 5g of phosphotungstic acid dissolved in 2mol/L hydrochloric acid and added to 100ml.

4. phenol reagent: in 1500ml spherical flask with sodium tungstate 100g, sodium aluminate (Na2MoO4-2H20) 25g, 700ml of water, 50ml of concentrated phosphoric acid, concentrated hydrochloric acid 100ml: slowly with a fine stream of distillation for 10h. Remove the condenser tube, add lithium sulphate 75g, 50ml of distilled water and add 2-3 drops of bromine, and then boiled! 15min, to remove excess bromine, cooled and diluted to 1000ml, made of phenol reagent is bright yellow, placed in a brown bottle to save, take out a part before use, with an equal amount of distilled water to dilute it.

5. 1.88 mol / L sodium carbonate solution.

6. standard tyrosine solution (1ml = 0.05mg); accurately weigh 5mg of tyrosine AA, 0.lmol / L hydrochloric acid solution and diluted to 100ml.

II. experimental operation:

Serum 0.5 ml, add 154 mmo / L sodium chloride 4.5 ml, mix well, 1.8 mol / L, perchloric acid solution 2.5 ml, rest for 10 min, with quantitative filter paper filtration or centrifugation. Take 2.5ml of the solution, add 17.74mmol / L phosphotungstic acid 0.5ml mixing, rest for 10min, centrifuged at 3000r / min for 10min, pour off the supernatant drained, and then add 2ml of phosphotungstic acid solution, suspension of precipitates, centrifuged in the same way, pour off the supernatant. Take the precipitate as standby.

Determination according to the following table


Mixing, placed in a 37 ℃ water bath for 15min, take out the colorimetric, spectrophotometer 650nm, colorimetric cup aperture 1.0cm, with a blank tube to adjust zero, read the absorbance of each tube.

III. Calculation:

Serum mucin (g / L) = assay tube absorbance / standard tube absorbance × 1.785 serum mucin (mg / L) (tyrosine) assay absorbance / standard tube absorbance × 75

Reference value: 0.71-0.87g/L for protein.

Tyrosine: 33.8-27mg/L

Caveat

1. This method has many influencing factors. Normal value observation reports vary, so whether the results are expressed in protein or casein AA, the method used and the normal value must be described.

2， the operation of serum and 154mmol / L sodium chloride solution dilution must be accurate.

3、The speed of titrating perchloric acid should be slow. It is appropriate to add 2.5ml in 30-40s, the speed is too fast, turbidity is easy to occur, and it is not easy to get a clear supernatant after centrifugation.

4， perchloric acid precipitation of serum protein at 30 ℃ below the better, otherwise the results of low.

5、Perchloric acid is a strong oxidizing agent, it should be stored according to the regulations of dangerous goods.

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