Liquid B-gal assays

Helen McBride helen_mcbride at hlthsci.med.utah.edu
Tue Dec 17 16:42:42 EST 1996


Hi Mike,
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. 

!-Galactosidase Assay
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
sample.  

Protein Determination
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)
extract.  

4.   1.0 OD595 = 18.9 !g using BSA standards.  

Calculations: 
Open !ONPG Macro 1.10! and follow directions for data entry. The macro
uses these formuli.
A)  mg/ml protein  =    OD595  x!(18.9)
                                        !l assayed
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
7/29/94 WV
Recipes:
1X Z Buffer:
0.06M   Na2HPO4 
0.04M   NaH2PO4 
0.01M   KCl  			
0.001M  MgSO4 	 
0.05M   !-mercaptoethanol
 -For 100ml:
   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

ONPG Solution:
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 
(prepare fresh)

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
-from Bio-Rad
 
ONPG: o-Nitrophenyl-!-D-Galactopyranoside
-from Sigma   

Bradford Reagent
Helen McBride
University of Utah 
Grad Student
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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