Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Medium 670
Day 2
24 hours
Friday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day 5
96 hours
Monday Renew the HepaRG® Thawing/Plating/General Purpose Medium 670
Day
Tide Quencher™ 5
(TQ5) as a quencher and iFluor™ 670 as a
fluorescent donor on the N- and C- terminals
respectively where the fluorescence of iFluor 670
is effectively quenched by TQ5 when the peptide
light.
6. Measure the absorbance at 670 nm (A670).
These assays can be adapted for use in standard 1 mL
cuvettes. To adapt to cuvettes follow protocol as for
96 well plates but scale up volumes as
concentration (nM) on enzyme
activity is shown in Figure 2.
Figure 2.
Inhibition of BACE1 activity by a specific inhibitor,
[Asn
670
, Sta
671
, Val
672
]-Amyloid /A4 Precursor Protein
6. Measure the absorbance at 670 nm (A670).
These assays can be adapted for use in Standard 1 mL
cuvettes. To adapt to cuvettes, follow protocol as for
96 well plates but scale up
into a “V”
bottomed 96-well plate. (see guava manual for instrument compatible plates) NOTE: Plate
must be placed on ice before adding cells.
12. Centrifuge cells at 670 x g for 5 minutes in a
Measurement
Record absorbance at 670 nm (A670) in end
point mode.
Results
1. Subtract the 0 Standard A670 reading
from Standards A670 readings.
2. Plot the Standard curve with A670 on
into a “V”
bottomed 96-well plate. (see guava manual for instrument compatible plates) NOTE: Plate
must be placed on ice before adding cells.
15. Centrifuge cells at 670 x g for 5 minutes in a
containing 13 mL.
Materials Not Supplied
1. Test tubes for sample preparation and storage
2. 96-well, v-bottom plate (Costar: 3897). *Recommended but not required if performing assay in a
plate
into a “V”
bottomed 96-well plate. (see guava manual for instrument compatible plates) NOTE: Plate
must be placed on ice before adding cells.
17. Centrifuge cells at 670 x g for 5 minutes in a
into a “V”
bottomed 96-well plate. (see guava manual for instrument compatible plates) NOTE: Plate
must be placed on ice before adding cells.
17. Centrifuge cells at 670 x g for 5 minutes in a
oxidation-
specific epitopes mediate macrophage recognition. Proc Natl
Acad Sci U S A. 1999 96: 6353-8.
2. Competitive ELISA Protocol
To test the specificity of the E06-TopFluor™ binding to antigens
plasma samples 2-fold with water.
Procedures
96 well plate Assay
1. Transfer 5 µL of diluted standards, Blank, and
diluted samples to appropriate wells of a clear
bottom plate.
2. Add 200 µL of
cells for 5
minutes at 670 x g between each wash.
31. Resuspend cells in 0.5 mL of ice cold 1X Assay Buffer.
32. Analyze 200 µL (approximately 300 to 500 cells/µL for 96 well
samples 2-fold with water.
Procedures
96 well plate Assay
1. Transfer 5 µL of diluted Standards, Blank, and
diluted Samples to appropriate wells of a clear
bottom plate.
a fluorescence spectrometer), or in a 96-well microplate
with glass-bottom (read-out on Laser-Scanner or fluorescence microplate reader).
It is applicable for 2 different protein concentration ranges
Buffer. Incubate cells for 20 minutes on ice.
• Remove fixation buffer by centrifugation at 670 x g for 5 minutes. Aspirate and discard
supernatant, only saving the cell pellet.
• For every