Preparation of Tissue Sample for Western Blotting

 

 

Important: Keep tissue cold, i.e. perform all steps on ice.

 

1.         Collect  tissue and snap-freeze in liquid  nitrogen.  Store at -70^o C.

 

2.         Weigh tissue sample.  Mince tissue with clean razor blade.

 

3.         Transfer tissue to a centrifuge tube and add 2-3 mL of 2.5 M Tris-HCl

Buffer (pH 7.2) to tube.   Homogenize tissue.

 

4.         Centrifuge @ 6000 rpm for 15 min (at 0^o C).

 

5.         Transfer supernatant to a clean tube; put perforated cap on tube or

cover opening of tube with parafilm and poke holes in it.

Freeze at  -70^o C (~ 20 min.).   Lyophilize overnight.

 

 

Protein Assay

 

1.         Dilute 200 uL of Stock BSA Standard (1.5  ug/uL) with 1800 uL

ddH2O (working conc:  0.15  ug/uL).

 

2.         Dilute stock dye with ddH₂O in the ratio of 2 parts dye to 1 part ddH₂O. Mix.

(Stock Dye--> Bio-Rad Protein Assay Dye Reagent Concentrate)

 

3.         Prepare standards and reagent blank as follows (in duplicate):

 

 

Working Std             ddH2O                       Dye

Reagent Blank                           0.0 uL                     1000.0 uL                   400 uL

5 Std                                         35.5 uL                     964.5 uL                    400 uL

10 Std                           71.0 uL                     929.0 uL                    400 uL

15 Std                         106.5 uL                     893.5 uL                    400 uL

20 Std                         142.0 uL                     858.0 uL                    400 uL

25 Std                         177.5 uL                     822.5 uL                    400 uL

 

t                  Add protein and then ddH₂O, mix.  Add dye, mix.

 

4.         Add 1.5 mL ddH₂O to lyophilized sample ( on ice).

 

5.         Take 6  uL of that and add 236 uL ddH₂O (1/40 dilution).

 

6.         Take 100 uL of that and add 900 uL ddH₂O and 400 uL of dye (1/14 dilution).

Do in duplicate.

  

7.         Use Spectrophotometer to obtain Absorbance values.  Read at 595 nm.

Use water to zero, then use reagent blank to re-zero.

 

8.         Average the two readings obtained for each standard and for the sample.

 

9.         Do linear regression analysis using Excel.  This will give you the

value of the y-intercept (Intercept/Coefficients) and the slope

(X-variable 1/Coefficients).

 

Please Note:  x (Independent variable) = Concentration of Standards

y (Dependent variable)   =  Absorbance of Standards

 

Conc. of Standards = (Vol. of BSA working Std)  (Conc. of BSA working Std)

Total Volume

 

e.g.     [5 Std] = 35.5 uL ( 0.15 ug/uL)   =  0.00380 ug/uL

                        1400 uL

 

 

t                   5  Std             0.00380 ug/uL

t                  10 Std             0.00761 ug/uL

t                  15 Std             0.01141 ug/uL

t                  20 Std             0.01521 ug/uL

t                  25 Std             0.01902 ug/uL

 

The above standards will be appropriate for samples with a concentration

between 2.13 ug/uL and 10.65 ug/uL (determined by multiplying the

concentration of the lowest and highest standard by 560).  If  the concentration

of your sample is outside these limits, make up additional standards

and repeat the absorbance readings.

 

10.       Using the formula   x= (y – b)/m ,  calculate the concentration of your

sample.   This formula is a variation of the classic  y= mx + b,

where,  in this case,  x = concentration (ug/uL)

                                    y = absorbance

                                    m = slope

b = y-intercept

The value you obtain for x will be the concentration of a  1/560

dilution of the sample.   Therefore, multiply this value by 560 to

obtain the correct concentration.

 

If the concentration of the sample is too low,  you may freeze it at -70^o C,

re-lyophilize it, and then reconstitute sample in a smaller volume of ddH₂O.

If this is the case, repeat the protein assay.   Store samples at -70^o C.

 

11.       To calculate the total amount of protein obtained from tissue:

 

Total  Protein = (Sample conc.) (reciprocal of dilution factor) (total vol. of sample)

 

* dilution factor = 1/40 (1/14) = 1/ 560        reciprocal = 560

*  total volume of sample = 1500 uL  (1.5 mL ddH₂O were added to

 lyophilized sample)