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Western Blot (BioRad Apparatus)

1.) Run SDS-Gel as usual per protocol.

2.) After run separate plates and isolate gel. Soak for 15min. in Tank Buffer without SDS.

Tank Buffer: 7.5 gm Tris-base
36 gm Glycine
500 ml Methanol
2.5 g SDS (or 25 ml 10%) add later
-----------------------------------------------------
2500 ml H₂O

3.) Soak Nitrocellulose membrane in ddH₂O until wet.

4.) Set up blotting-sandwich: positive red electrode (clear side)

=======

sponge (soak in tank buffer + SDS)
3 sheets 3mm Whatman (soak in tank buffer + SDS)
nitrocellulose membrane
gel
3 sheets 3mm Whatman soak in tank buffer =======
negative black electrode (black side)

roll off air bubbles with pipette on membrane and Whatman.

5.) Put sandwich into apparatus and add little stirring bar to bottom. Fill chamber with Tank Buffer + SDS and blot for 2 hours at 200 mA at RT, with stirring. (For the mini-Western apparatus add the ice pack insert and run at 100V (150 mA) for 1 hr)

6.) Then take off membrane, mark the gel position on the filter, and rinse in TBS (10 mM Tris, 0.14 M NaCL pH 7.5) 3x at RT for 1 minute each, with gently shaking.

7.) Incubate membrane in Blotto (5% instant milk in TBS) for 2 hours at RT or O/N at 4 C

9.) Dilute 1st Ab in blotto (1/100 or 1/5000) and incubate membrane in seal -a-meal bags for 2 hours at RT or O/N at 4 C.

(25 ul of 1:400 prediluted Cappel rabbit anti-bgal ab in 10 ml Blotto, 1 hr, RT)

10.) Rinse 3x in TBS for 15min.

11.) Dilute 2nd Ab (peroxidase conjugated G mouse IgG (H+L); use rabbit to detect protein A).

1:500 in blotto. Incubate with membrane for 1 - 2 hours at RT.

( for chemiluminescence-2.5 ul BRL goat anti rabbit IgG ab, horse radish peroxidase conjugate in 10 ml Blotto, 1 hr, RT)

12.) Rinse with TBS 3x for 15min.

13.) Develop in fresh 60 mg 4-chloro-1-naphtol in 20 ml methanol, add 80 ml 10 mM Tris pH 7.5 and 50 ul 30% H₂O2.

or for chemoluminescence assay

2.5 ml Amersham ECL reagent 1 (RPN-2106)
2.5 ml Amersham ECL reagent 2
5 ml TBS

14.) Immerse membrane or for chemiluminescence wrap in Saran wrap and perform autoradiography (approx. 15 second exposure).

 

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