research: BCA assay

it's week 16! how's everyone doing!

this week, i'm going to explain what is BCA protein assay.

what is BCA protein assay? it is an assay that quantitates protein concentration.

what does BCA stands for? BCA stands for bicinchorinic acid.

what is the principle of this assay?

principle of this assay:

2 major reactions occur in this assay

1. proteins are held together by peptide bonds. upon exposure to copper ions (Cu2+), the peptide bonds will reduce Cu2+ to Cu+. this reduction process is temperature dependent. this also means that, the more proteins you have in the solution, the more copper ions are reduced to Cu+.

2. BCA molecules will bind to Cu+, resulting in the formation of a purple-coloured product. the colour intensity of this product is then measured at wavelength 562nm using a plate reader. (colourimetic assay)

how does the assay work?

the assay usually comes in a kit. inside the kit, you can find: BCA reagents A and B, 96-well flat bottom plate, Bovine . Albumin (BSA) standards.

BSA standards: as the name suggests, BSA is a serum albumin, and we all know that albumin is a protein. so the BSA standards serve as a control for the kit (standard control). the BSA concentration that the company provides is usually 2mg/ml (2000ug/ul). to get a series of concentrations, we dilute the BSA (from high concentration to low).

Range of concentration of BSA diluted: from 0mg/ml to 2mg/ml. usually we can have up to 6-7 standards inclusive of 0mg/ml. BSA is diluted in ultrapure water (Milli-Q water).

Eg,

well A: 0 ug/ml (no BSA standard) /BLANK

well B: 131.072 ug/ml BSA

well C: 262.144 ug/ml BSA

well D: 327.68 ug/ml BSA

well E: 409.6 ug/ml BSA

well F:512 ug/ml BSA

well G: 640 ug/ml BSA

well H: 2000 ug/ml BSA

next, we prepare the BCA working reagents .

BCA working reagents = Reagent A + Reagent B (in ratio 50:!)

reagent A is colourless, reagent B is blue in colour.

one important thing to note is that the working reagent must be prepared freshly prior to use and stored at 4 degrees celsius. Do not keep overnight.

upon adding reagent B, the solution immediately turns GREEN. mix well.

the next component we will require is THE BUFFER. this buffer, simply refers to the the buffer we use in our samples. eg, if the samples are cells, then the buffer is the cell culture medium.

steps:

1. Add 20 ul of BSA standards to the individual wells.

2. Add x ul of sample to each well.

3. Add x ul of buffer to the wells that contain BSA standards.

4. Add 20 ul of Milli-water to the wells that contain samples.

Steps 3 and 4: This is to eliminate disturbances caused by any possible absorption by the buffer when measuring the absorbance of samples. Similar reason applies to adding 20uL of water into the samples’ wells.

5. Add (200-20-x) ul of BCA working reagents to all the wells.

- therefore total volume in each well: 200 ul.

-upon adding, you will find that the solution turns purple in the presence of proteins

6. Mix well, by pipetting up and down.

7. Cover the plate.

8. Incubate at 37 degrees celsius for 30 minutes.

- before reading on the plate reader, ensure that there are no bubbles. the presence of bubbles will affect the reading.

9. Read at wavelength 562 nm.

Simple? Yeah.

Any alternatives?

some of you may have used before the bradford assay. For those who have not, bradford assay is an alternative to BCA protein assay. (Read here for more information on bradford assay (wiki) : http://en.wikipedia.org/wiki/Bradford_protein_assay )

BCA protein assay is better than bradford as:

1. more sensitive compared to bradford assay

that's all! (:

LIM JIA HUI (JOEY)

0703605f tg01

group 2