Submitted by Lauren French (download pdf)

NPY ASSAY


NPY PURIFICATION:

Preparation of Reagents:

Solutions:
Acetonitrile (100%)
Buffer A:  1% TFA in 18 MΩ H20
(Add 10 mL TFA to 990 mL 18 MΩ H2O)
Buffer B:  60% acetonitrile and 1% TFA in 18 MΩ H2O
(Add 120 mL acetonitrile and 2 mL TFA to 78 mL 18 MΩ H2O)
Columns:
Peninsula Laboratories Inc
Cat # Y-1000; SEP Columns packed with C18 sorbent

Test tubes:
FisherBrand 12 x 75 mm polystyrene test tubes
Cat # 14-961-10A

Procedure:

1)    Set up columns and test tubes in racks.  Be sure to include a column for a standard NPYconcentration.Do not place columns above tubes until eluting the sample with buffer B.
2)    Prepare standard NPY solution (~2 - 5 ng/mL recommended) in same buffer as samples were prepared.  (This can be done using the "Standard Serial Dilutions" from the NPY ELISA protocol.)
3)    Wash columns with 100% acetonitrile.  Be sure all solution is down to column sorbent prior to the next step.
4)    Add 3 mL Buffer A.  Repeat two more times.  After 3rd Buffer A wash, be sure all solution is down to the column sorbent before next step.
5)    Load samples onto columns.  Be sure all solution is down to column sorbent prior to proceeding with Buffer A washes.
6)    Run 3 mL Buffer A through column.  Repeat two more times.  After 3rd Buffer A wash, be sure all solution is down to column sorbent. 
7)    Place columns over test tubes.
8)    Add 3 mL Buffer B to elute NPY sample.  This should be collected in the test tubes. 
9)    After all solution has been collected in the tubes, dry the samples in a speed vac overnight.  [Ifplanning on running NPY ELISA the next day, be sure to complete NPY ELISA protocol up throughstep4.]


NPY ELISA

Preparation of Reagents:


Buffers: (store at 4oC)

Assay Buffer:                                                                        Wash Buffer:
1.42 g Na­2HPO4 (dibasic)                                                1.42 g Na2HPO4 (dibasic)
0.2 g KH2PO4                                                                        0.2 g KH2PO4
1 mL Tween 20                                                                     1 mL Tween 20
Bring to 1 L, pH 7.4                                                            Bring to 1 L, pH 7.4
(500 mg BSA + 100 mL buffer made day of use)

Coating Buffer:                                                            Blocking Buffer:
4.3 g NaHCO3                                                                      8.0 g NaCl
5.3 g Na2CO3                                                                       1.42 g NaH2PO4 (monobasic)
Bring to 1 L, pH 9.4                                                               0.2 g KH2PO4
0.2 g KCl

Bring to 1 L, pH 7.4
(250 mg BSA + 50 mL buffer made dayof use)

 

Plate:
Corning Costar (Cat #3361) EIA/RIA plate
NPY antibody:
Rabbit Anti-NPY, Bachem Cat #T4070.  Do NOT follow reconstitution directions on data sheet.  Reconstitute the 50 uL lyophilized serum
in 500 uL 18 MΩ H2O.  Separate into 10 uL aliquots and storeat -20oC

Biotinylated NPY (Bio-NPY):
Bachem Cat #H-5674n Reconstitute 0.5 mg in 500 uL 18 MΩ H2O (1 mg/mL).  Store in 10 uLaliquotsas Bio-NPY Stocksolution at -20oC.
Streptavidin-HRP (SA-HRP):
BD Pharmingen Cat #554066Store at 4oC.
 
TMB:
1 Step Ultra TMB-ELISA, Pierce Cat #34028 Store at 4oC.

2N H2SO4

NPY Standards:
Bachem Cat #H-6375
Reconstitute to 1 mg/mL with 18 MΩ H2O.  Store at 4oC.

 

Procedure:

Preparations to be done the day prior to the experiment:

1)    Make all buffers as discussed above and store at 4oC.  Prepare all lyophilized reagents as above, ifhaven't already done so.

2)    Make coating buffer/anti-NPY antibody solution (1:25,000 dilution):  add 10 uL anti-NPY antibody(aliquots stored at -20oC) to 24.99 mL of coating buffer in 50 mL conical tube. Vortex.

3)    Obtain 96 well EIA/RIA plate

4)    Add 100 uL of coating buffer/anti-NPY antibody solution to each well.  Cover plate with plastic cover andincubate overnight at 4oC.

 

Day of Experiment


5)    Fill out a plate map, leaving 2 wells for non-specific binding (NSB) and 2 wells for total binding (TB). No blank necessary

6)    Mix 500 mg BSA with assay buffer to 100 mL total.  This is the working assay buffer to be used for making standards and throughout the remainder of the experiment.

7)    Reconstitute NPY samples in 200 uL assay buffer.  Vortex.

8)    Remove plate from refrigerator and wash 1X with 300 uL/well of wash buffer.

9)    Mix 250 mg BSA with blocking buffer to 50 mL total.  Add 300 uL to each well and incubate for 1 hour.

10) During incubation with blocking buffer, make the standards as explained in the table below.  Also make any sample dilutions with assay buffer during this time.

Start with NPY Standard stock solution (4oC). Make standards as follows:

Dilute stock 1:10 in assay buffer

(10 uL NPY standard stock + 90 uL assay buffer = Std A)

Dilute Std A 1:100 in assay buffer

(10 uL Std A + 990 uL assay buffer = Std B)

 

 

 

11) After 1 hr, wash plate 3X with 300 uL wash buffer per well.  Shake plate over sink to remove solution after each wash.  After final wash, invert plate over a paper towel and tap to remove remaining wash buffer.

12) Load plate.  Pipette 100 uL assay buffer into NSB and TB wells.  Add 100 uL of standards or samples into designated wells (as set up on the plate map) and incubate for 1 hour.

13) Carefully shake solution from plate over the sink.  Invert plate over a paper towel and tap gently to remove remaining solution. (DO NOT WASH)

14) Obtain 10uL aliquot of Bio-NPY stock solution (stored at -20oC).  Dilute as follows.

Dilute Stock 1:10 in assay buffer

(10 uL stock + 90 uL buffer = Bio-NPY A)

[Aliquot remaining Bio-NPY A into 10 uL aliquots and store at -20oC]

Dilute  Bio-NPY A 1:100 in assay buffer

(10 uL Bio-NPY A + 990 uL buffer à  Bio-NPY B)

Dilute Bio-NPY C 1:500 in assay buffeR

(20 uL Bio-NPY B + 9980 uL buffer à  Bio-NPY C)

 

15) Add 100 uL Bio-NPY C to all wells EXCEPT NSB.  Cover plate with plastic cover and incubate 2 hours atroom temperature.

16) Wash plate 3X as in step 7 above.


17) Make SA-HRP solution by adding 1 uL SA-HRP to 10 mL assay buffer in a 15 mL conical tube.  Vortex. Add 100 uL to each well.  Cover plate and incubate for 1 hour.

18) Wash plate 3X as in step 7 above.


19) Add 100 uL TMB to each well, cover plate, and incubate for 5-10 minutes.  Look for a strong blue color inthe TB wells.

20) Stop the reaction by addition of 100 uL 2N H2SO4 to each well.  Be sure there are no bubbles in thewells.  Read plate in plate reader at 450 nm within 30 minutes. 

 

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