Section 10- Protein BCA
Sample Type: choices are Reference, Standards 1-7, and Sample. The software will guide you to measure your
reference, then at least one standard before allowing measurement of samples. Reference samples refer to the dye
reagent without any protein added (ie the “0” sample).
Replicate #: counter for tracking replicate number during reference and standard measurement.
Reset all Standards (F11): clears all replicates of all standards.
Reset 1 Standard (F12): clears all replicates of the selected standard.
Absorbance at 562nm: the Cu-BCA complex’s absorbance at 562 nm for the 1mm pathlength.
Cursor λ and Absorbance: this feature allows the user to adjust the cursor wavelength and view the corresponding
absorbance. The cursor wavelength can be set by the user. Note: The user-selected wavelength and absorbance are not
utilized in any calculations.
mg/ml: the concentration of the sample (unknown).
Making BCA Measurements
A standard curve is required every time the BCA assay is run. Although curves can be saved and reloaded in the
NanoDrop
®
ND-1000 Spectrophotometer software, it is recommended that the user follow manufacturers’ guidelines and
generate fresh standard curves for each assay. Additionally, a standard curve ‘set-up’ may be reloaded. This feature will
recall the respective standard series used in a previously saved standard curve. Both single and multi-point standard
curve generation is incorporated into the software. A standard curve can be developed using a reference (BCA reagent
only – no protein) and a single replicate of one standard. The multi-point standard curve generator allows a maximum of
five replicates for each of seven different standards. There is no set order in which standards must be run.
A blank must be measured before the standard curve may be generated. It is advisable to use water as the blank and use
the dye reagent without any protein added as the “0” or reference sample. There are three general procedural steps to
unknown protein concentration measurements. The requisite order, including generating the standard curve, is as follows:
Step 1: Measure the
‘Reference’ (BCA
reagent – a ‘zero’
Standard)
Note: The software
will guide the user
with instructions in the
large text box on the
right side of the
screen.
10-2
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