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my western blots of membrane protein do not work - (Feb/25/2010 )

Hi,
I made 2 western blot with the same protocol, I can't see my protein of interest, membrane protein.
By ECL developping, no signal in the film. after, 5, 15 and 1h of exposure.
I suspect that the protein extract is not transfered to the nitro.membrane, I used semi-dry transfer apparatus.
I added DNase to remove DNA from my samplesand i kept on 37C for 5min, after i boiled for 5min and load 50ug. I can't see the protein by western!!!!!! I used RIPA +protease inhibitor cocktail to solublise the proteins in the sample.
If you have any suggestion. please reply me!!
thanks

-luciana-

Could you please post a little more about your procedure?

Some ideas:

1. Is the transfer buffer made correctly?
2. Did you place the gel on the right side of the membrane for transfer?
3. When you coomassie-stain the gel post-transfer, are any bands left that match the MW of your protein? (looking for complete transfer)
4. What voltage and time are you using?
5. Are you using an appropriately conjugated antibody?

-lab rat-

too many things could have gone wrong. check every step.
in addition to other suggestions, do you have a positive control to verify that the antibodies are working. either of the antibodies could have gone wrong. ECL might have degraded or not working.

-scolix-

the transfer buffer i used is tris- glycine1X+methanol 20%
the gel was placed in the right position, for coomassie staining, I couldn't see proteins a part from the bands of the marker.the transfer took 1h30 at 9V!
the antibody is goat HRP.
thanks for your interaction
if you have any advice

-luciana-

in fact, i was thinking if the protein extract was degradated!!! by puting with Dnase at 37C!!
since i tried one incubation with another antibody of actine, just to check if there are proteins in my sample. but i couldn't see the band of actine after ECL!!!!
so, im wondering what is the step in which the total extract was degradated.
i solubilise on ripa, on ice, sonicate, centrifuge at 4c, dnase at 37c, add loading bufer,boil and load!! rrun, the gel,

-luciana-

luciana on Feb 25 2010, 02:25 PM said:

the transfer buffer i used is tris- glycine1X+methanol 20%
the gel was placed in the right position, for coomassie staining, I couldn't see proteins a part from the bands of the marker.the transfer took 1h30 at 9V!
the antibody is goat HRP.
thanks for your interaction
if you have any advice



You can't see protein after coomassie? Do you have access to silver stain or Sypro Ruby stains? Are you lysing enough cells? Instead of using DNase, you could pass the lysate through a needle a few times. You could skip the DNase step to see if that improves yield.

-fishdoc-

luciana on Feb 25 2010, 03:40 PM said:

in fact, i was thinking if the protein extract was degradated!!! by puting with Dnase at 37C!!
since i tried one incubation with another antibody of actine, just to check if there are proteins in my sample. but i couldn't see the band of actine after ECL!!!!
so, im wondering what is the step in which the total extract was degradated.
i solubilise on ripa, on ice, sonicate, centrifuge at 4c, dnase at 37c, add loading bufer,boil and load!! rrun, the gel,


Proteins are not degraded by putting the sample at 37 deg C and DNase should not affect the proteins.
You should check that protein is transferred if you have a stain for the membrane (e.g., Ponceau), but make sure that you use a new blot if any of the steps already done would interfere with the stain (check the staining procedure protocol to determine this).
You should also check that the ECL is still working. Sometimes it simply goes bad if unused for a while, left at room temp for too long, or if the two reagents were inadvertently mixed at one point. It would be best to borrow some ECL, and re-test your antibody of interest, followed by another control (anti-actin, etc.). If still negative, you can then stain the membrane with Ponceau (see above) or another stain to make sure the transfer is working.

-amelia417-

thanks for your answers, in fact i made another western last week. Without using DNAs, I homogenized the cells with one apparatus for lysis for eppendorf, then lysis by sonication. I used blotting appartus for 1 hour instead of semi-dry. I could see proteins by rouge ponceau staining!! However, I could'nt see my protein of interest by western!!
i suspect that the primary antibody is not working, I bought it 4 months ago and used several times!!!
I' m going to use another one bought the same time as the first but not yet opened!!!
In the datasheet, this antibody (Santa Cruz company) is stable for one year at 4C!!
if you have any idea.
thanks alot

-luciana-

I have problem with my western too, I hav recobint protein tried western for that. I tried all the protocol whic discuss in your forum, did not get any band. At the last I am thinking my primary is not working

-ALQ-

You should test your primary with easier techniques, such as serial dilutions of the lysate spotted onto a membrane, followed by attempts at detection. Only when that is working at the detection levels you care about should you bother with gels and transfers.

-phage434-