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Western blot- bands weak

western PVDF primary blocking

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10 replies to this topic

#1 sdheeru

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Posted 28 February 2013 - 09:07 PM

I am having some trouble with western blotting. I follow same protocol as my fellow lab mate (as following)

2x sample buffer with 5% BME
PVDF membrane
0.05% tween in TBST
5% milk for blocking, primary and secondary.

He always gets single sharp bands for all the antibodies he tries (without any background). Even though I follow the same protocol, I either get VERY weak bands or background/multiple bands appears.

I have used his sample buffer, gel making buffer, same antibody concentration but nothing seems to help.

Also I get 2 bands for a mAB which is not supposed to be there.

I certainly can't match his experience but I somehow need some help to get my experiments moving.

Suggestions wanted.

#2 aimikins

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Posted 28 February 2013 - 11:43 PM

so what is the thing that is different in your westerns (compared to your labmates)? is it the protein preparation? perhaps your samples have less target protein and you need to start with more? are your samples possibly being degraded?

can you talk about your sample preparation method for the loading protein?
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#3 science noob

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Posted 01 March 2013 - 01:25 AM

Did you use the sample total protein sample? a-ha

weak bands -
1) check your loading control (actin, tubulin, gapdh, histone h3) - are they weak as well
2) how much total protein sample did you load?

background -
1) did you use the same membrane? PVDF tends to gather more background than nitrocellulose
2) check your secondary antibody dilution

multiple bands -
1) are you probing for the same protein expression as your labmate?
2) some antibodies (yes, even monoclonal) give you multiple bands (non-specific, multiple isoforms) - what protein are you probing for?

#4 sdheeru

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Posted 01 March 2013 - 09:35 AM

so what is the thing that is different in your westerns (compared to your labmates)? is it the protein preparation? perhaps your samples have less target protein and you need to start with more? are your samples possibly being degraded?

can you talk about your sample preparation method for the loading protein?


The samples are basically the same. I have prepared the samples in RIPA buffer (with PI cocktail and phosphatase inhibitors) and he used the same sample with same amount of protein loading.

I just can't figure out what could be going wrong.

#5 sdheeru

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Posted 01 March 2013 - 09:42 AM

Did you use the sample total protein sample? a-ha

weak bands -
1) check your loading control (actin, tubulin, gapdh, histone h3) - are they weak as well
2) how much total protein sample did you load?

background -
1) did you use the same membrane? PVDF tends to gather more background than nitrocellulose
2) check your secondary antibody dilution

multiple bands -
1) are you probing for the same protein expression as your labmate?
2) some antibodies (yes, even monoclonal) give you multiple bands (non-specific, multiple isoforms) - what protein are you probing for?


We use only the PVDF membrane in our lab. Do you think it could have anything to do with the wetting the membrane. He usually keeps it in the transfer buffer as long as the gel is running. While I have usually keep it for 15-20 min in the transfer buffer ( before starting with the gel transfer).

The proteins and concentrations are also the same.

#6 mdfenko

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Posted 01 March 2013 - 12:06 PM

do you (or does he) first wet the membrane in methanol (then transfer to transfer buffer)?
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#7 sdheeru

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Posted 01 March 2013 - 02:31 PM

do you (or does he) first wet the membrane in methanol (then transfer to transfer buffer)?


yeah for 5 min in methanol and then in transfer buffer.

#8 mdfenko

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Posted 01 March 2013 - 02:40 PM

do you set up the transfer the same way as your labmate?

do you ensure contact between the gel and membrane before transfer?

do you use the same antibody?

if using ecl, do you react the reagent for the same amount of time (from introduction through exposure, for the same exposure time)?
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#9 sdheeru

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Posted 01 March 2013 - 04:04 PM

do you set up the transfer the same way as your labmate?

do you ensure contact between the gel and membrane before transfer?

do you use the same antibody?

if using ecl, do you react the reagent for the same amount of time (from introduction through exposure, for the same exposure time)?


Transfer is for same and same voltage.

I always make sure the gel and membrane have no bubbles in between.

The antibodies are same.

Earlier I used to put ECL for 1 min and then keep the membrane in the cassette. Then I tried for 30 s as he does, I don't see any improvement (either bands are weak or bg is high).

Possible things I think could affect are:

-The cassette for developing the blot. He has white cassette (from inside) and mine is black.

-PVDF wetting time.

-The side of gel used against the membrane and ultimately upside for the antibody solutions.

Do you think these factors could be a cause??

#10 science noob

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Posted 01 March 2013 - 04:24 PM

Earlier I used to put ECL for 1 min and then keep the membrane in the cassette. Then I tried for 30 s as he does, I don't see any improvement (either bands are weak or bg is high).


What do you mean you keep the membrane in the cassette? How did you probe your membranes with the primary & secondary antibodies - I do mine in small clip-lock plastic food containers. Same question would also go with how did you wash your membranes - in the cassette?



-The cassette for developing the blot. He has white cassette (from inside) and mine is black.

-PVDF wetting time.

-The side of gel used against the membrane and ultimately upside for the antibody solutions.

Do you think these factors could be a cause??


Does 'developing' mean transfer?

A rule of thumb, the sequence of your transfer 'sandwich' cassette is:
black side (-), sponge/fiber pad 1, blotting paper 1, gel, membrane, filter paper 2, sponge/fiber pad 2, white side (+). So when you open your cassette after a transfer, your membrane will be on the white cassette.

Check the nice picture to represent what I described here: http://www.abcam.com...ource&rid=13045


QUESTION:
1. Did you run any protein ladder/markers?
2. What was your protein of interest?
3. Did you probe the same membrane for housekeeping proteins/loading controls - e.g. actin, tubulin, GAPDH, histone H3?

#11 mdfenko

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Posted 04 March 2013 - 06:06 AM

Possible things I think could affect are:

-The cassette for developing the blot. He has white cassette (from inside) and mine is black.

possible. white would reflect light, black will absorb. try his white cassette.

-the PVDF wetting time.

not likely, 30 seconds in methanol is more than sufficient to activate the membrane (i think 10 seconds is sufficient)

-The side of gel used against the membrane and ultimately upside for the antibody solutions.

not the side of the gel (both are equivalent) but the side of the blot that you are exposing.

have you determined how well the transfer went (stained the blot with ponceau and the gel with coomassie or silver after transfer)?
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