Liquid B-gal assays
helen_mcbride at hlthsci.med.utah.edu
Tue Dec 17 16:42:42 EST 1996
This is our liquid b-gal assay. We do use a glass bead lysis method and
it works great. We have a modified protocol for doing the assay in 96well
plates if your interested and have a temperature controlled plate reader.
We also have an Excel Macro which sets up the template with all the
calculations. E-mail me if you want that. Good Luck.
Quantitative Yeast !-Galactosidase Assay
Preparation of Extract;
1.Determine the appropriate amount of a stationary culture to add to 10
mls of media (in large culture tubes) so that after ~18 hrs of growth the
OD660 of the cultures will be ~1.0 (eg.- for wt strains innoculate with
2!l stationary culture). Label each tube with small piece of tape.
2. Spin down cells 1500 R.P.M. for 10 min. Pour off supernatant.
3. Add 1 ml Z buffer, vortex, and transfer into eppendorf tube. Transfer
the small tape to the eppendorf tubes.
4. Spin 30 sec in microfuge. Pour off supernatant.
5. Resuspend in 200 !l Z buffer. Add one measure of glass beads.
6. Vortex in tube shaker for 5 min (in cold room).
7. Spin 5 min in microfuge (in cold room).
8. Put tubes on ice until assayed.
1. Reactions are most easily done in disposable cuvettes. For each point
use one cuvette containing 1 ml Z buffer + 0.7 mg/ml ONPG. Prewarm the
cuvettes in a 28!C water bath.
2. Add 5-50 !l of extract to the cuvette with ONPG and Z buffer. Record
time of reaction start.
3. Observe the appearance of a light yellow color, at which point remove
cuvette, add 0.3 ml 1M Na2CO3, and record the time.
4. Measure the OD420. Linear range = 0.1< OD420 < 0.35. (One can wait
until all reactions are completed and read all of the OD's at once.)
5. Use a blank that has been incubated for the longest time used for any
1. Pipette 1 ml diluted Bradford reagent (1 part stock + 4 parts water;
prepared fresh) into disposable cuvettes. (Exercise caution -- Bradford
reagent contains phosphoric acid.)
2. Add 2-20 !l of extract. Mix gently. After 5 min. read OD595.
3. The linear range of the Bradford Assay is OD595 = 0.1-0.6. If your
sample is above or below these limits, then repeat using more (or less)
4. 1.0 OD595 = 18.9 !g using BSA standards.
Open !ONPG Macro 1.10! and follow directions for data entry. The macro
uses these formuli.
A) mg/ml protein = OD595 x!(18.9)
B) !-gal units = 1000 x OD420
(ml assayed) x (min assayed) x
(protein conc [mg/ml])
1X Z buffer + 0.7 mg/ml ONPG for multiple reactions:
1 rxn 20 rxns 25 rxns 30 rxns 50 rxns
!-mercaptoethanol 5!l 100!l 125!l 150!l 250!l
10X Z Buffer (no !-ME) 100!l 2ml 2.5ml 3ml 5ml
4 mg/ml ONPG 175!l 3.5ml 4.4ml 5.2ml 8.75ml
H2O 720!l 14.4ml 18ml 21.6ml 36ml
David Stillman 5/5/87; revised 2/17/88; 11/19/90(Pam); slightly revised
1X Z Buffer:
10 ml 10X Z Buffer w/out !-ME
350 !l 14.3M !-mercaptoethanol
-H2O to 100 ml
-Store at 4!C
10X Z Buffer w/out !-ME:
8.5 g Na2HPO4
5.52 g NaH2PO4!H2O
0.75 g KCl
1 ml MgSO4 (1M)
-Add H2O to 100 ml
4 mg/ml ONPG:
-Make up in 0.1M NaXHXPO4 pH=7.0
39 ml 0.1M NaH2PO4
61 ml 0.1M Na2HPO4
100 ml H2O pH=7.0
Diluted Bradford Reagent:
1 part stock
4 parts water
Glass Beads: 0.45-0.5mm from Sigma
-Cleaned for Reuse by washing in nitric acid, rinsing extensively in
ddH2O, then baking.
Cuvettes: 1.5 ml Semi-Micro Polystyrene
University of Utah
Dept. of Onc. Sci.
helen.mcbride at genetics.utah.edu
"Where the telescope ends, the microscope begins, which of the two has
the grander view?" Victor Hugo
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