Determination of protein concentration by BCA method

Summary

Determination of protein concentration in samples

Principle

BCA (bicinchonininc acid) and divalent copper ions of copper sulfate and other reagents composed of reagents, mixed together to become apple green, that is, BCA working reagent. Under alkaline conditions, BCA and protein binding, the protein will Cu2 + reduced to Cu +, a Cu + chelate two BCA molecules , the working reagent from the original apple green to form a purple complex, the maximum light-absorbing intensity is proportional to the protein concentration.

Appliance

biological research

Operation method

BCA protein quantification

Materials and Instruments

Reagents
BSA, protein standard solution, PBS, reagent A (1% BCA, disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% sodium hydroxide, 0.95% sodium bicarbonate, mixed to adjust the PH value to 11.25), reagent B (4% copper sulfate);
Equipment
Spectrophotometer

Move

1. Preparation of protein standard

Prepare 25mg/ml protein standard solution: 30mg BSA in 1.2ml protein standard preparation solution, stored at -20℃. Prepare 1ml of 0.5mg/ml protein standard solution and store at -20℃.

25mg/ml Protein Standard Solution	PBS
20ul	980ul

1. Preparation of BCA working solution

BCA reagent (A:B) = 50:1, mix well and stabilize at room temperature for 24h.

1. Standard curve was drawn ( 20ul system)

0.5mg/ml BCA(ul)	0.5mg/ml BCA (ul)	0.5mg/ml BCA(ul) 0 1	2	4	8	12	16	20
PBS	20	19	18	16	12	8	4	0
Protein (ug)	0	0.5	1	2	4	6	8	10

• Add 200ul of BCA working solution to each well, mix well and place at 37℃ for 20-30min.

(Note: It can also be left at room temperature for 2 hours or at 60ºC for 30 minutes.) The color of the BCA assay will deepen over time as the protein concentration is measured. The color will increase with time and the color reaction will be accelerated by increasing temperature. If the concentration is low, incubate at a higher temperature or for a longer period of time).

• Colorimetric measurement at 562nm
• Plot the standard curve with absorbance value as the horizontal coordinate and protein content (ug) as the vertical coordinate.

1. Protein concentration detection
   • Add 200ul of BCA working solution to each well, add 20ul of dilution solution (1ul of sample to be tested + 19ul of PBS) to each well.
   • Mix well and place at 37℃ for 20-30min.
   • Colorimetric measurement at 562nm
   • The corresponding protein content (ug) can be found on the standard curve.

Caveat

If you inoculate the same density of cells, similar treatment conditions, and the number of cells is basically the same, then you can directly use SDS loading buffer to lyse for 30 min, and then run WB with an unlimited number of samples. it is recommended to take photos with Rachidon red staining as a record.

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