Filename: enzassay doc Written by: J. Ciccotosto (revised 3/8/98) Protocol: phm and pal assays



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Filename:ENZASSAY.doc Written by: J. Ciccotosto (revised 3/8/98)

Protocol: PHM and PAL Assays
I PHM Assay

Making HOT mix (stock)

Stock.

Final conc.

125I-Ac-Tyr-Val-Gly




20K cpm/tube

Ac-Tyr-Val-Gly

100 µM

0.5 µM

Catalase

20mg/ml

0.1mg/ml

CuSO4 (may need to optimize)

100µM

0.5 µM

Ascorbate (make fresh stock in water, daily)

50 mM

0.5 µM

NaMES (150 mM, pH 5.0)

* bring to volume


II PAL Assay

Making HOT mix.

Stock.

Final conc.

125I-Ac-Tyr-Val-OH-Gly




6 Kcpm/tube

Ac-Tyr-Val-OH-Gly

100 µM

0.5 µM

Lubrol (thesit)

10%

0.02%

NaMES (150 mM, pH 5.5)

* bring to volume


III General Notes

Assay samples in duplicate tubes

Add <1 µg protein per tube (If cell extracts have 2 mg/ml, make a dilution so that you add less than 0.5 µl)

Per assay include (in duplicate), add 4 total tubes, 2 positive control tubes, and 2 blank tubes.

NaOH is added to drive all available substrate through to the end point.

Tris-HCl stops reaction.


IV Calculation Of Results

refer to PHM assay template for excel spread sheet program.




Chemical

Vendor

Catalog #

Ascorbate

Sigma

A-0278

Catalase

Boehringer Mannheim

106810

Cupric Sulphate (CuSO4)

Sigma

C-6283

Sodium Hydroxide (NaOH)

J.T. Baker

3722-05

Thesit

Boehringer Mannheim

1332520

Trizma Base (C4H11NO3)

Sigma

T-1503


PHM ASSAY COVER SHEET

Assay Date: Assay Number:

Experiment:

Number of samples = __________

Total volume = 40 l x __________ samples = __________l

Reaction Volume = __ l x __________ = __________l



HOT MIX

label (125I-Ac-YVG) = µl

cold Ac-YVG (100 M stock; 0.5 M final) /200 = µl
Catalase (20 mg/ml stock; 0.1 mg/ml final) /200 = µl
CuSO4 (100 M stock; 0.5 M final) /200 = µl
Ascorbate (50 mM stock; 0.5 mM final) /100 = l

Total Vol = µl

NaMES (150 mM, pH 5.0) = µl


Time Start: __________ Time Stop: __________ Total Time: __________

PHM assay procedures

  1. Make sure water bath (in Rm 905) is ON, set at 37 oC and with enough water in it.

  2. NOTE: In making HOT MIX, add Ascorbate last, just before starting assay.

  3. Make sample dilutions in diluent buffer. Aliquot out samples into microfuge tubes.

  4. In each assay, include 4 RIA tubes for total cpm counts , 2 mf tubes for blank (background counts) and include QC samples.

  5. Add hot mix to ALL tubes, incubate in the 37oC water bath for at least 1 hour.

  6. After incubation, spin tubes for 1 min at 3000 rpm.

  7. Add 15 µl NaOH (1 N) and incubate (at RT) for 4 min (including blanks).

  8. Add 240 µl blue Tris/HCl (1M, pH 7.0)

  9. Do Ethyl Acetate steps in fume hood in Rm 905. Dispose of old ethyl Acetate in fume hood in Rm 902 sink. Add 640 µl of fresh water-saturated ethyl-acetate. Cap tube and shake vigorously.

  10. Be sure cap is ALL the way on the tube before putting on gamma counter.

  11. Take 320 µl off the top phase and place into RIA tube and cap. Count each sample for 2 min.


PAL ASSAY COVER SHEET
Assay Date: Assay Number:

Experiment:_________________________________________________________________

Number of samples = __________

Total volume = 40 l x __________ samples = __________l

Reaction Volume = __ l x __________ = __________l
HOT MIX

label (125I-Ac-YV-OH-G) = µl



cold Ac-Tyr-Val-OH-Gly (90 M stock; 0.5 M final) _____/180 = µl

Lubrol (Thesit; 10% stock; 0.02% final) _____/500 = µl

Total Vol. = µl

NaMES (150 mM, pH 5.5) = l


Time Start: __________ Time Stop: __________ Total Time: __________
PAL assay procedures

  1. Make sure water bath (in Rm 905) is ON, set at 37 oC and with enough water in it

  2. Make sample dilutions in diluent buffer. Aliquot out samples into microfuge tubes.

  3. In each assay, include 4 RIA tubes for TOTAL cpm counts , 2 mf tubes for BLANK (background counts) 2 mf tubes for BASE counts and include QC samples .

  4. Add hot mix to tube and incubate in a 37oC water bath for at least 1 hour.

  5. After incubation, spin tubes for 1 min at 3000 rpm. DO NOT ADD NaOH.

  6. Add 240 µl blue Tris/HCl (1M, pH 7.0)

  7. Do Ethyl Acetate steps in fume hood in Rm 905. Dispose of old ethyl Acetate in fume hood in Rm 902 sink. Add 640 µl of fresh water-saturated ethyl-acetate. Cap tube and shake vigorously.

  8. Take 320 µl off the top phase and place into RIA tube and cap. Count sample for 2 min.

NOTE: Include duplicate BASE tubes to which 15 µl NaOH (1N) is added;incubate for 4 min.


Eipper/Mains Protocol Manual


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