Protocols · Benchling

Materials

96-well plate
P₂₀₀ & P₂₀₀ Sterile Tips
P₂₀ & P₂₀ Sterile Tips
BCA Reagent A
- sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1M sodium hydroxide

BCA Reagent B
- 4% cupric sulfate
BSA 2mg/mL
Microtubes
TBS-CHAPS 2%
Protein samples

Preparation of Diluted BSA Standards

Label 7 microtubes of 0.6mL from 0 to 7. These will be for your BSA standards.

Compute the final concentration in each microtube.

7	N/A	280 µL BSA	µg/mL
6	140 µL MQ dH2O	140 µL BSA Std. 7	µg/mL
5	140 µL MQ dH2O	140 µL BSA Std. 6	µg/mL
4	140 µL MQ dH2O	140 µL BSA Std. 5	µg/mL
3	140 µL MQ dH2O	140 µL BSA Std. 4	µg/mL
2	140 µL MQ dH2O	140 µL BSA Std. 3	µg/mL
1	140 µL MQ dH2O	140 µL BSA Std. 2	µg/mL
0	240 µL MQ dH2O	N/A	µg/mL

Label microtubes for your protein sample dilutions ( dilutions = 1:2, 1:4, 1:8; Cyto = cytosolic; Mito = mitochondrial ) and blanks (Bk).

Note: Blank by definition is everything except the sample (just the buffer). Therefore, Bk Cyto is blank for the cytosol fraction is a mix of all the reagents A & C from the mitochondrial isolation kit that were mixed to obtain the cytosolic fraction (1.25 parts Reagent A and 1 part Reagent C), while Bk Mito is just the TBS-CHAPS 2%.

Bk Cyto	Cyto 1:2	Cyto 1:4	Cyto 1:8
Bk Mito	Mito 1:2	Mito 1:4	Mito 1:8
Bk Prot	Prot 1:2	Prot 1:4	Prot 1:8

Bk Cyto	312.5 μL Reagent A Mitochondrial Extraction Kit	250 μL Reagent C Mitochondrial Extraction Kit
Cyto 1:2	100 μL Bk Cyto (Reagents A & C Mix)	100 μL Pure Cytosolic Fraction
Cyto 1:4	100 μL Bk Cyto (Reagents A & C Mix)	100 μL Cytosolic Fraction 1:2
Cyto 1:8	100 μL Bk Cyto (Reagents A & C Mix)	100 μL Cytosolic Fraction 1:4

Bk Mito	150 μL TBS-CHAPS 2%	N/A
Mito 1:2	40 μL TBS-CHAPS 2%	40 μL Pure Mitochondrial Fraction
Mito 1:4	40 μL TBS-CHAPS 2%	40 μL Mitochondrial Fraction 1:2
Mito 1:8	40 μL TBS-CHAPS 2%	40 μL Mitochondrial Fraction 1:4

Bk Prot	150 µL Lysis buffer	NA
Prot 1:2	40 μL Lysis Buffer	40 μL Total Protein
Prot 1:4	40 μL Lysis Buffer	40 μL Protein 1:2
Prot 1:8	40 μL Lysis Buffer	40 μL Protein 1:4

Mix the ingredients according to Tables.

Preparation of the BCA Working Reagent (BCA WR)

Analyze the total amount of wells (Figure 1 or 2) that you are going to use during the laboratory experience.

(1) Multiply the total amount of well that are needed by 200 μL (total volume of BCA WR).

(2) Then modify this volume to prepare a little bit more as contingency.

Use the following formula to determine the total volume of BCA WR required.

Total Volume BCA WR Required = Total # of Wells in the Microplate x Volume of BCA WR per Sample

Determine the amount of BCA Reagent B to be mixed with the volume of BCA Reagent A calculated.

WR is prepared at a dilution ratio of 1mL Reagent B per 50mL BCA Reagent A

Mix the volume of BCA Reagent A & BCA Reagent B in a test tube.

Note: When BCA Reagent B is mixed with BCA Reagent A, turbidity is observed that quickly disappears upon mixing to yield a clear, green BCA WR.

Microplate Procedure

Add protein dilutions to microplane wells

Cytosolic Fraction: Pipette 25 μL of each standard, unknown samples and appropriate blanks replicates into a microplate well.

Mitochondrial Fraction: Pipette 20 μL of each standard, unknown samples and appropriate blanks replicates into a microplate well.

Total protein samples: Pipette 20 μL of each standard, unknown samples and appropriate blanks replicates into a microplate well.

Add 200 μL of the BCA WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.

Cover plate and incubate at 37°C for 45 minutes.

00:45:00

Cool plate to Room Temperature.

Measure the absorbance at or near 562 nm on a plate reader.

Construct the Standard Curve for Total Protein Determination in the Cytosolic and Mitochondrial Fractions