Section 12- Protein Bradford
Reset Window (F11):
clears all replicates of all standards.
Reset This Std (F12):
clears all replicates of the selected standard.
Absorbance at 595nm:
protein-dye complex’s absorbance at 595 nm at 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.
ug/ml:
concentration of the sample (unknown).
Show Report:
formatted for 200 samples although the buffer size can be increased to accommodate thousands of samples.
Making Bradford Protein Measurements
A standard curve is required every time the Bradford 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 (Modified Lowry 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 measurement. The requisite order, including
generating the standard curve, is as follows:
Step 1:
Measure the ‘Reference’
(Bradford reagent – a ‘zero’
Standard)
The software will not allow
measurement of samples until at
least 1 standard and reference – 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
Sample
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 of the data. [Note:
In order to obtain a concentration
value (ug/ml) the sample (unknown)
must fall within the limits of the
standard curve.
12-2
