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Western not working anymore


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15 replies to this topic

#1 cm13

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Posted 22 July 2010 - 04:17 AM

I performed a western blot that didnt work.

I got the gel working ok, and Ponceau staining showed that the transfer worked perfectly.

I blocked the membrane in 5% BSA in 0.1% TBS-Tween for 1 hour.
Then i washed it for 5 minutes in 6mls of 0.1% TBS-Tween. I did this 3 times.
I then added my primary antibody.

I had gotten the antibody working before at 1:1000 concentration in 5% BSA.
So i was trying this again.
I also tried 1:2000 and 1:5000.
The primary was left overnight at 4C.

The next day:
I washed it for 5 minutes in 6mls of 0.1% TBS-Tween. I did this 3 times.
I then added my secondary antibody.
They were at (1:1000, 1:2000 and 1:5000 concentrations also, matching the primary)
I left the blots shaking at room temperature for an hour.
I washed it for 5 minutes in 6mls of 0.1% TBS-Tween. I did this 3 times.
Then i developed it.

The result wasnt good.
The blots appeared completely Black in colour.
And on one of them (the 1:5000) i could see some banding, but it was wildly non-specific.
It was as if the antibody had acted upon the whole amount of protein.

Where am i going wrong with all of this?

#2 WYF

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Posted 27 July 2010 - 12:41 AM

I think either the blocking step had failed or the concentration of the antibody is too high. If the whole blot turned out black, it's more likely the secondary that's too high i think...

#3 cm13

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Posted 27 July 2010 - 03:02 AM

I think either the blocking step had failed or the concentration of the antibody is too high. If the whole blot turned out black, it's more likely the secondary that's too high i think...


Are you sure about the secondary? Like i said, i was doing low concentrations (1/5000) of it on one blot.
It turned out mostly black too. There was a patch where i could see some bands, but they were non-specific.

I also checked my TBS-T ph just to make sure there wasnt a problem there. It's ph 7.2, but should have been 7.6 so i brought it up to that, and now im trying that.
Im thinking maybe reducing the BSA to a lower concentration as that may be my problem to begin with.

#4 Prep!

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Posted 27 July 2010 - 03:13 AM

i wont say 5000 is a huge dilution so u can stop thinking abt it.. i use my secondary in a 15000 dilution!!!
i am confused as to why u use only 6 ml to wash your blots!!!! i wud overflow the blot with washing solution for the said incubation times!!!
i think ur blocking is fine.. i too use 5% skimmed milk to block!!!
i dont lnopw how useful this will be but i generally dont wash the blot between blocking and primary!!!

Edited by Prep!, 27 July 2010 - 03:15 AM.

Support bacteria - They are the only culture some people have!!!
Cheers!!!

#5 sera_tonin

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Posted 27 July 2010 - 10:35 AM

i would guess that the secondary antibody is too concentrated - i usually use 1:1000 primary and 1:10,000 secondary, sometimes 1:20,000. also, try using more wash buffer in your washes; i use as much will fit in my container without spilling, which is at least 50ml - the whole point of the wash is too dilute as much excess antibody as possible and wash it away.

good luck!

#6 bob1

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Posted 28 July 2010 - 01:14 PM

I agree on too high a secondary concentration. YOu could also have a shorter 2ry incubation time, increase your wash volume and do extra washes - up to 5 per antibody incubation.

#7 cm13

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Posted 29 July 2010 - 12:44 AM

Hello again.
So i retried my westerns as before, but this time i washed 4 times for 6 minutes in TBST where needed.
I did see an improvement in the clearness of the blots.
I also ran a range of different protein concentrations (5, 10, 20 and 30ug per well) just to see if the amount i was adding made a difference.

The blots appeared clearer after the extra washings.
With the blots where i did a 1/2000 or 1/5000 dilution of the Primary antibody, nothing appeared.
With the blots with a a 1/1000 Primary concentration, there were bands, but they were non-specific.
They were stronger (specific and non-specific) bands in the 20 and 30ug wells, but that would be expected.

Im going to try a 1/1000 of primary and a 1/5000 or 1/10000 of secondary next, but does anyone have any other suggestions of what to do to avoid non-specific binding?

#8 bob1

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Posted 29 July 2010 - 02:15 PM

Try adding Tween or Triton to your primary antibody diluent, you could also try 1:1200, 1:1500, etc. and incubating at room temp or in the fridge for a shorter time, it might help a bit.

#9 cm13

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Posted 29 July 2010 - 11:20 PM

Well, the TBS already has 0.1% Tween in it. I figured this would be enough?

#10 sera_tonin

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Posted 30 July 2010 - 11:51 AM

Hello again.
So i retried my westerns as before, but this time i washed 4 times for 6 minutes in TBST where needed.
I did see an improvement in the clearness of the blots.
I also ran a range of different protein concentrations (5, 10, 20 and 30ug per well) just to see if the amount i was adding made a difference.

The blots appeared clearer after the extra washings.
With the blots where i did a 1/2000 or 1/5000 dilution of the Primary antibody, nothing appeared.
With the blots with a a 1/1000 Primary concentration, there were bands, but they were non-specific.
They were stronger (specific and non-specific) bands in the 20 and 30ug wells, but that would be expected.

Im going to try a 1/1000 of primary and a 1/5000 or 1/10000 of secondary next, but does anyone have any other suggestions of what to do to avoid non-specific binding?


nonspecific bands are usually from too much primary antibody, in my experience - but if you're not getting bands at a higher dilution, then you don't have much choice. it could just be that this particular antibody has nonspecific binding - have you used it before without getting the extra bands?

#11 cm13

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Posted 30 July 2010 - 11:56 AM


Hello again.
So i retried my westerns as before, but this time i washed 4 times for 6 minutes in TBST where needed.
I did see an improvement in the clearness of the blots.
I also ran a range of different protein concentrations (5, 10, 20 and 30ug per well) just to see if the amount i was adding made a difference.

The blots appeared clearer after the extra washings.
With the blots where i did a 1/2000 or 1/5000 dilution of the Primary antibody, nothing appeared.
With the blots with a a 1/1000 Primary concentration, there were bands, but they were non-specific.
They were stronger (specific and non-specific) bands in the 20 and 30ug wells, but that would be expected.

Im going to try a 1/1000 of primary and a 1/5000 or 1/10000 of secondary next, but does anyone have any other suggestions of what to do to avoid non-specific binding?


nonspecific bands are usually from too much primary antibody, in my experience - but if you're not getting bands at a higher dilution, then you don't have much choice. it could just be that this particular antibody has nonspecific binding - have you used it before without getting the extra bands?



Yes. I tried it before and it got specific bands. But to repeat the experiment a while later gave me non-specific ones.
I thought it could have been because i re-use antibodies a couple times, but even with fresh antibody, the same thing happened.

Could this mean i have a problem maybe with my cell lysates?

Edited by cm13, 30 July 2010 - 11:57 AM.


#12 bob1

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Posted 01 August 2010 - 02:15 PM

How is your antibody stock stored? If it is in the fridge, then it may be going off. All our antibodies get stored aliquotted at -20 and are only thawed once to prevent degradation. I have come across antibodies that degrade very fast (less than a week in the fridge).

#13 cm13

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Posted 02 August 2010 - 05:33 AM

How is your antibody stock stored? If it is in the fridge, then it may be going off. All our antibodies get stored aliquotted at -20 and are only thawed once to prevent degradation. I have come across antibodies that degrade very fast (less than a week in the fridge).



I freeze it in its diluted (in BSA) form at -20C.
I also only ever re-use the antibody 3 times at most, as it would go off afterwards.

#14 sera_tonin

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Posted 02 August 2010 - 10:47 AM

Yes. I tried it before and it got specific bands. But to repeat the experiment a while later gave me non-specific ones.
I thought it could have been because i re-use antibodies a couple times, but even with fresh antibody, the same thing happened.

Could this mean i have a problem maybe with my cell lysates?


did you prepare the tissue the same way as before? it could be something like, this time you solubilized more membrane proteins than last time, and some of those membrane proteins are binding the antibody. i don't know :huh: sometimes the biochemistry gods conspire to throw a monkey wrench into our experiments, for no logical reason...

#15 cm13

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Posted 26 August 2010 - 03:55 AM

Hi again everyone.

At the recommendation of others, i performed westerns with the following concentrations:

Primary 0, Secondary 1:1000
Primary 1:1000, Secondary 1:1000
Primary 1:2000, Secondary 1:1000


The results were as follows:

Primary 0, Secondary 1:1000 - Bands appeared as before - as if the antibody attached to a lot of proteins
Primary 1:1000, Secondary 1:1000 - Same as the blot with no primary, but with some bands being stronger.
Primary 1:2000, Secondary 1:1000 - No bands at all.

Surely this indicates that im having a problem with my Secondary?
Should i try 1:1000 for primary and then lower concentrations for the Secondary?




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