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Western Blot

Transfer buffer with methanol and SDS

1x transfer buffer          100 ml of 10 x Transfer buffer

                                   700 ml dH2O

20% methanol              200 ml of Methanol

chill at 4° while electrophoresing

Acrylamide Gel Buffers

Upper Gel Buffer:          0.5 M Tris Base                        15.15g

                                   0.4% SDS                                1.0g

                                   pH 6.8                                            add H2O to 230 ml, pH to

                                                                                         6.8, addmore H2O to 250 ml

                                                                                         (takes a lot of concentrated HCL)

Lower Gel Buffer:          1.5M Tris Base                          90.9g

                                   0.4% SDS                                2.0g

                                   pH 8.8                                           add H2O to 460ml, pH with concentrated

                                                                                        HCI to 8.8 (takes alto) and add more H2O

                                                                                        to 500 ml. 

Acrylamide solution:     Acrylamide                                73g

                                   Bis Acrylamide                          2g

                                   (30% @ 37.5:1)                         add H2O to 250 ml

1) Electrophoresis

Add 0.1 A280 units/well (1 A280 unit is 1 ml of lysate at an A280 reading of 1) Add 10 µl Broad molecular weight markers and color molecular weight markers. Electrophorese @ 150-200 V

2) Blotting Wet PVDF membrance 15 sec. in methanol, then 3 min. in dH2O, then 3 min, in transfer buffer. Blot 1 hr. for proteins <50 kd to 2 hr for proteins> 180 kd at 4° in 1x transfer buffer. For large proteins, add SDS to transfer buffer (0.1% final concentration).

3) Disassemble blotting apparatus and dry blot at 42° for at leat 1 hr or room temp overnight. 

4) Incubate blot in TBST until translucent grey (~15m). Pour off TBST and add 2% milk / TBST and mix 30 min. Pour off blocking buffer. 

5) 1° antibody Dilute antibody into enough 0.2% skim milk / TBST to cover blot (usually 20ml). monoclonal antibodies are usually diluted 1:2000 - 1:10000. Polyclonal antibodies are usually diluted about 1:50000.

Incubate 1 hr. at room temp. 

6) Pour of antibody solution and wash 3 x 15 sec. with TBST, then 3x 5 min. in TBST.

7) 2° antibody Mix blot 1 hr. 0.2% skim milk / TBST with 1:50000 dilution HRP conjugated 2° antibody.

8) Wash as in step 15. 

9) Add 1 ml of each part of chemiluminescent reagents together in a 2 ml microfuge tube. Lay a clean piece of transparency plastic on top of a saran wrap, then distribute about a dozen drops of chemiluminescent reagent to the plastic. Lay the blot onto the drops and add the rest of the reagent to the top of the blot. Overlay another piece of transparency plastic applying even, gently pressure to squeeze out excess reagent, then spread the excess with gloved fingers back over the top of the transparency "sandwich" (the liquid will help the saran wrap to adhere more evenly. Fold the saran wrap over the transparency, then fold the edges of the sran wrap back to prevent any reagent leaking out. 

The transparency plastic is used to ensure even contact of the chemiluminescent reagent with the blot. 

10) Expose to film or chemiimager for 5 min. to 2 hours. Pierece "Supersignal Fempto" chemiluminescent reagent works much better on the chemiimager than the "Supersignal Pico" "Fempto has a much longer stronger signal, but as a consequence can have higher background and is much more expensive.