Solarbio Albumin Content Assay Kit (Bromocresol Green Colorimetry)

產品概述

Product Components

溶液製備:

1. Color Development Solution:
   • Prepare the color development solution according to the number of samples by mixing Reagent One, 試劑二, and Reagent Three in the ratio of 10μL: 990微升: 10微升 (1010μL for 5 tests). Mix thoroughly and use immediately.
2. Standard Solution:
   • 準備一個 10 mg/mL albumin standard solution. 使用前, take 200μL of the 10 mg/mL albumin standard solution and add 200μL of the extraction solution to prepare a 5 mg/mL albumin standard solution. Mix thoroughly and use immediately.

產品描述:

• Albumin:
  • The liver synthesizes the main protein in human plasma.
  • An important nutrient that maintains plasma osmotic pressure.
  • Binds with various nutrients, hormones, and drugs.
  • Reflects the nutritional status of the body.
  • Used to screen for diseases affecting liver metabolic function (例如, cirrhosis, liver damage, malnutrition, malignant tumors).
• Detection Method:
  • In a pH 4.2 buffer solution, serum albumin carries a positive charge.
  • In the presence of a non-ionic surfactant, albumin binds with the negatively charged dye Bromocresol Green.
  • Forms a blue-green complex.
  • The complex has an absorption peak at a wavelength of 630 奈米.
  • Color intensity is directly proportional to the concentration of albumin.
• Reaction:
  • Albumin + Bromocresol Green → Blue-Green Complex (630 奈米)

筆記:

• Before the experiment, selecting 2-3 samples with expected large differences is recommended to conduct a preliminary experiment.
• If the sample absorbance is not within the measurement range, diluting or increasing the sample volume is recommended for detection.

Required Instruments and Supplies (to be prepared):

• Visible spectrophotometer/microplate reader
• Low-temperature centrifuge
• 分析天平
• Microglass cuvettes/96-well plates
• Adjustable pipettes
• Mortar/homogenizer/ultrasonic cell disruptor
• Ice and distilled water

程式:

樣品加工 (The sample volume can be adjusted accordingly; specific ratios can be referenced from literature):

1. Tissue Samples:
   • Add extraction solution in a ratio of sample mass (G) 至萃取液體積 (毫升) 的 1:5〜10 (recommend weighing 0.1g of the sample and adding 1.0mL of extraction solution).
   • Homogenize the sample in an ice bath, then centrifuge at 4℃, 8000克為 10 分分鐘.
   • Discard the pellet and keep the supernatant on ice for testing.
2. Bacterial/Cell Samples:
   • Add extraction solution in a ratio of bacterial/cell count (10^6) 至萃取液體積 (毫升) of 5~10:1 (recommend adding 1.0mL of extraction solution to 5 million bacteria/cells).
   • Disrupt the bacteria/cells in an ice bath using an ultrasonic cell disruptor (power 200W, ultrasonicate for 3s, 間隔7秒, 總時間 5 分分鐘).
   • 4℃離心, 8000克為 10 分分鐘.
   • Discard the pellet and keep the supernatant on ice for testing.
3. Liquid Samples:
   • Measure directly.
   • If the liquid is turbid, centrifuge and use the supernatant for measurement.

1. Preparation of Visible Spectrophotometer/Microplate Reader:
   • Preheat the visible spectrophotometer/microplate reader for at least 30 分分鐘.
   • Set the wavelength to 630 奈米.
   • Zero the visible spectrophotometer with distilled water.
2. Operation Table (Add the following reagents to micro glass cuvettes/96-well plates):

• Mix well and let stand at room temperature for 20 秒.
• Measure the absorbance of each tube at 630 奈米, recording the values as A_test, A_standard, and A_blank.
• Calculate ΔA_test = A_test – A_blank, and ΔA_standard = A_standard – A_blank.
• Note that the blank tube and standard tube only need to be measured 1-2 次.

筆記: The length of the standing time can affect the detection results. It is recommended to react directly in the micro glass cuvettes/96-well plates for 20 seconds and then measure the absorbance.

Albumin Content Calculation

1. Calculation by Sample Protein Concentration

Albumin content (毫克/毫克蛋白質) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ (V sample × Cpr) = 5 × ΔA measured ÷ ΔA standard ÷ Cpr

2. Calculation by Sample Mass

Albumin content (mg/g mass) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ (W × V sample ÷ V sample total) = 5 × ΔA measured ÷ ΔA standard ÷ W

3. Calculation by Bacterial/Cell Number

Albumin content (mg/10^6 cells) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ (V sample × N ÷ V sample total) = 5 × ΔA measured ÷ ΔA standard ÷ N

4. Calculation by Liquid Volume

Albumin content (毫克/毫升) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ V sample = 5 × ΔA measured ÷ ΔA standard

Definitions:

• C standard: Standard tube concentration, 5 毫克/毫升;
• V sample: Sample volume added, 0.02毫升;
• V sample total: Total extract volume added, 1毫升;
• 心肺復甦: 樣品蛋白濃度, 毫克/毫升;
• 瓦: Sample mass, G;
• 氮: Total number of bacteria/cells, in 10^6

筆記:

1. If ΔA_test is less than 0.010 or the absorbance of the test tube is close to that of the blank tube, you can increase the sample volume before measurement. If ΔA_test is greater than 0.5, it is recommended to appropriately dilute the sample supernatant with extraction solution before measurement. Note to simultaneously adjust the calculation formula.
2. If the sample becomes turbid after adding the color developer, it is recommended to appropriately dilute the sample supernatant with extraction solution before measurement. Note to simultaneously adjust the calculation formula.

Experimental Example:

1. Take 20μL of human serum sample, dilute it 10 times with extraction solution, follow the measurement steps, and measure it using a 96-well plate. 計算: ΔA_test = A_test – A_blank = 0.426 – 0.124 = 0.302, ΔA_standard = A_standard – A_blank = 0.406 – 0.124 = 0.282. Calculated based on liquid volume: Albumin Content (毫克/毫升) = 5 × ΔA_test ÷ ΔA_standard × 10 (dilution factor) = 53.546 毫克/毫升.