Application

This kit can be used for detection of the total protein in serum, plasma, tissue, culture cells and other samples. 

Detection principle

Cu2+ can be reduced to Cu+ by protein under alkaline conditions. Cu+ can combine with BCA reagent and form purple complex, which has a maximum absorption peak at 562 nm. The OD value is proportional to the protein concentration. The protein concentration can be calculated indirectly according to the OD value at 562 nm.

Experimental instrument

Tube, Micropipette, Vortex mixer, 37℃ water bath, Spectrophotometer (562 nm)

Sample pretreatment

1. Animal tissue homogenate: Weigh the tissue accurately. Add normal saline according to the ratio of Weight (g): Volume (mL) =1:9. Mechanically homogenize the sample in ice water bath and centrifuge at 2500 rpm for 10 min. Take the supernatant and dilute with normal saline to prepare 1% or 0.5% tissue homogenate for detection.

2. Plant tissue homogenate: Accurately weigh the tissue weight, add homogenized medium (0.1 mol/L, pH7.4 phosphate buffer or normal saline are recommended) according to the ratio of Weight (g): Volume (mL) =1:9. Mechanically homogenize the sample in ice water bath and centrifuge at 3500 rpm for 10 min. Take the supernatant and dilute with normal saline to prepare 1% tissue homogenate or 0.5% tissue homogenate for detection.

3. Serum (plasma) sample: Dilute the sample with normal saline at a ratio of 1:49 (or 1:99) for detection.

4.  Cells:

(1) Cell collection: Take the prepared cell suspension and centrifuge at 1000 rpm for 10 min. Discard the supernatant and keep the cell sediment. Wash the sediment with iso-osmia buffer (0.1 mol/L, pH7~7.4 phosphate buffer was recommended) 1~2 times, centrifuge at 1000 rpm for 10 min, discard the supernatant and keep the cell sediment.

(2) Cell disruption: Add homogenate medium (0.1 mol/L, pH7~7.4 phosphate buffer or saline was recommended). Sonicate in ice water bath (power: 300W, 5 second/time, interval for 30 sec, repeat for 3~5 times) or grind with hand-operated, then take the prepared homogenate sample for detection without centrifugation.

5. Other liquid sample: Dilute the sample with an appropriate dilution multiple. The detection range is 20~2000 μg/mL. It is recommended to take 1-2 samples for a preliminary experiment before the formal experiment.

Operation steps

Technical parameters

1. The linear range is 20~2000 μg/mL, R2=0.999.

2. The intra-assay CV is 2.06 % and the inter-assay CV is 4.72%.

3. The recovery of the kit is 98.5 %.

Product advantages

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of the kit is 6 months.

4. Do not use components from different batches of kit.

5. This kit is more convenient than the classical Lowry method. The coefficient of variation for detection of different protein is less than the Coomassie method

6. The minimum detection limitation is 20 μg/mL and the minimal detection amount of protein is 0.5 μg.

7. This kit will not be interfered by chemical substances which include ionic and nonionic detergent, and is compatible with 5% SDS, 5% Triton-X-100 and 5% Tween 20/60/80 in sample.

8. The container for preparation of reagents should be avoided of protein contamination due to the high sensitivity.