Section 10- Protein BCA
mg/ml:
the concentration of the sample (unknown).
Show Report:
formatted for 200 samples although the buffer size can be increased to accommodate thousands of samples.
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. 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.
There are only 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)
The software will not allow
measurement of samples until at
least 1 standard and references –
or 2 standards – are measured.
Note: Software will guide the user
with instructions in the large text
box on the right side of the screen.
The
red
light indicates the
standard curve is incomplete and
not yet ready for sample
measurements.
Step 2:
Measure Standards
Up to 5 replicates of each
standard can be measured. The
software will not allow
measurement of samples until a
minimum of 1 standard and
references – or 2 standards – are
measured. Polynomial curve fitting
requires more standard points
depending on the polynomial
degree selected
.
Step 3:
Measure Samples
Once a minimum standard curve
has been established, the
red
indicator light will turn
green
,
allowing the user to start
measuring samples. Sample
concentrations are calculated by
using linear interpolation (point-to-
point) between the two standards
flanking the unknown sample
or
by using polynomial fitting. [Note:
In order to obtain a concentration
value (mg/ml) the sample
(unknown) must fall within the
limits of the standard curve].
Standard Curve Features
Standard curves can be saved and reloaded for reference use by using the ‘Standard Curve’ pull down menu and choosing save or
reload functions. Selecting the ‘View Standard Curve’ button allows the user to review the standard curve at any time.
The Delete Point
button allows the user to delete points by first selecting the data point to be deleted in the table and then choosing the
‘Delete Point’ button. If erroneous or non-representative readings are encountered for a specific standard, all replicates of that standard
are cleared by selecting ‘Reset this std’. Additionally, all standards can be deleted at once using the ‘Reset This Window’ (F11) button.
10-2
