why I can not get clear specific bond of COX-2 - what wrong with my protocol? (Dec/11/2008 )

1: Place the cell culture dish on ice box and wash the cells with ice-cold PBS(-) for 3 times

2: asperate the PBS(-), then add ice-cold lysis buffer

3: scrape adherent cells off the dish using a cold plastic cell scraper then gently transfer the cell suspension into a pre-cooled microfuge tube

4: homogenize the cell lysate with handy sonic for 4 times 10 sec /time in icebath

5: keep the cell lysate for 30 mins at 4 degree centigrade

6: centrifuge in a microtrifuge at 2000 rpm for 5 mins at 4 degree centigrade, for reove nucleus

7: gently transfer the supernatant into a new microfuge tube then stored in -20 degree centigrade

Lysis buffer:
Name

Final concenration

HEPES
50m M

NaCl
137m M

MgCl2
1 mM

CaCl2
1 mM

Na4 P2 O7
10 mM

NaF
10 mM

EDTA
2 mM

Glycerol
10%

Vanadate
2 mM

Aprotinin
10ug/ml

PMSF
2mM

Glycerophosphate
25mM

Trion-100
1%



II. 10% SDS-PAGE:


III. Membrane transference:


IV. Ag-Ab reaction:

IV.1. Blocking:

Incubate the membrane in blocking buffer at 4℃ overnight.

OR at room temperature for 1 hour.

IV.2. 1st Ab reaction:

Change into certain volume fresh blocking buffer and add certain 20 times diluted 1st Ab(final concentration of anti-cox-2 (Santa Cruz)is 5000 times diluted) to obtain the modest working dilution. Incubate at room temperature for 1 hour with shaking.

IV.3. Wash:

Change into certain volume fresh blocking buffer and incubate at room temperature with shaking for 5 min. Repeat 2 more times.

IV.4. 2nd Ab reaction:

Change into certain volume fresh blocking buffer and 5000 times anti- rabbit add certain 20 times diluted 2nd Ab to obtain the modest working dilution. Incubate at room temperature for 1 hour with shaking.

IV.5. Wash:

Change into certain volume fresh blocking buffer and incubate at room temperature with shaking for 5 min. Repeat 2 more times.

IV.6. Change box:

Transfer the membrane into a new box containing certain fresh blocking buffer and incubate with shaking for 5 min.

IV.7. Wash:

Change into certain volume fresh blocking buffer and incubate at room temperature with shaking for 5 min. Repeat one more time.


V. Fluorescence detection:

V.1. ECL:

Immerse the membrane in A&B solution mixture (1/1, v/v) and perform exposure as soon as possible.

V.2. Exposure: 5min, 15min, 30min or overnight

V.3. Development: 1~2min

V.4. Wash: 1~2min

V.5. Fixation: 5~10min

V.6. Wash: 1~2min

Your protocol so far seems to be o.k., I have seen just two points which I would try to change. First one is your lysis time on ice. Instead of just 30 min I would give it at least one hour or more to lyse on ice, so your protein concentration might be better. How much protein do you load onto your gels?
Second point is the concentration and duration of your 1st antibody step. 1:5.000 seems to me very diluted, normally first antibodies are dilutes between 1:100 and 1:1000, try some points of dilution in this range on a small blot with just positive/negative controls to get the right ab concentration. And the incubation time of only 1h at RT may be too short, normal time is 2h at RT, try this! Or even better: Overnight at 4°C shaking.

Your exposure times are very long, normally your reaction should start after 2-3 min and hold on for max 15-30 min. I don't think that overnight exposure will give you better results than exposure for 30 min. But more important is the 1st antibody. Try to change the conditions. Good luck!