Bicinchoninic Acid (BCA) Protein

Assay

BM40012
 Learning Outcomes for today
• Understand the principle of the protein assay called BCA
• Understand how to make and why a standard curve is used
to measure protein content of an unknown sample
• Where this assay is used
• Why we use replicates in our assays
• What are appropriate controls
 What is a protein?

https://www.genome.gov/genetics-glossary/Protein
Why would we want to measure protein
in a clinical setting?
• Assess Kidney damage via inflammation (glomerulonephritis)
• Assess Kidney cancer
• Diagnosis of pre-eclampsia
• Assess dehydration
• Assess inflammation in diseases such as Lupus
• Assess Low Blood pressure
• Poisoning
• Assess Cardiovascular disease
• UTI
• Assess stress
Types of Tests to Measure protein
• Bicinchroninic acid (BCA)
• Lowry
• Bradford assay
• Amino acid assay
How are we measuring proteins today?
 How does the BSA assay work?
• Copper based colorimetric assay for measuring total proteins.
• It involves two reactions;
- Peptide bonds in the protein reduces Cu2+ to cuprous ions Cu+. The
amount of reduced copper is proportional to the amount of protein in
the sample.
- Colorimetric detection of the Cu+ ions by the BCA reagent. 2 BCA
molecules bind one Cu+ forming a purple BCA-Cu+ complex.
How does BCA work?
What are we doing today? – a microBCA

• Step 1: Make a standard Curve using Bovine Serum Albumin (BSA)

• Step 2: Prepare a working solution of BCA and copper sulphate (25:24:1 ratio)

+ +

• Step 3: Mix Standard curve proteins (0.15mls) with working solution (0.15mls)
and incubate for 30 minutes at 37oC in a 96 well plate

• Step 4: Cool to room temperature

• Step 5: Read samples in a plate reader
 Step 1: Making the standard curve

STOCK (2000pg/ml) DILUENT = H20

A = 200pg/ml
B = 40pg/ml
C = 20
D = 10
E=5
F = 2.5
G = 1.25
H =0.625
I-0 CONTROL
 Unknown Sample
• PATIENT’S URINE SAMPLE; you are checking for
proteinuria (high concentration of albumin protein in your
urine)
• CONTROL URINE
HOW MUCH WORKING SOLUTION DO WE NEED?
HOW MANY SAMPLES DO WE HAVE?
• NOS OF STANDARDS DO WE HAVE: 9 X 2 REPLICAS = 18

• NOS OF PATIENT SAMPLES DO WE HAVE: 1 X 2 REPLICAS = 2

• TOTAL SAMPLES BEING ADDED TO THE 96 WELL PLATE/GRP = 20

20 samples x 0.15ml = 3 ml
Step 2: Preparing a working solution (5ml)

+ +

PARTS 25 24 1

2.5ml 2.4ml 0.1ml (100uls)

0.25ml 0.24ml 0.1ml (10uls)

Which combination would you use?

Step 3: Mixing Standards/Sample with BCA

0.15ml sample/standard
+
0.15 ml of BCA
Health and Safety:

• Personal Protective Equipment (PPE): Always wear a lab coat, gloves, and safety
• glasses while performing the experiment.
• Biological Samples: Handle all biological samples with caution and dispose of them
• according to biohazard waste protocols established by your institution.
• BCA Working Solution: This solution is mildly irritating. Avoid contact with skin and eyes.
• In case of contact, flush immediately with plenty of water for at least 15 minutes. Seek
• medical attention if irritation persists.
• Sodium Hydroxide (NaOH): This is a corrosive chemical. Wear gloves and eye protection
• when handling. Dispose of waste according to proper chemical waste protocols. Consult the Safety Data
Sheet (SDS) for detailed handling and disposal information.
 Skills acquired today
• Pipetting and use of appropriate pipettes and tips
• Pipetting both small and large volumes
• Use of a spectrometer
• Calculations; ‘parts’
• Data analysis; ‘making a standard curve’
• Data Interpretation “how much protein do I have in my unknown
sample?”
• Teamwork
• PLEASE MAKE SURE YOUR SKILLS PASSPORT IS SIGNED
BEFORE LEAVING THE CLASS