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antibody solution in milk gettin sour fast

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  #1  
Old 09-30-2005, 12:44 PM
MMu
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Default antibody solution in milk gettin sour fast



Our primary antibody solutions are getting sour very fast even though we use
natrium azide and store at 4C at all times. Incubation is done at 4C
overnight as well.

1) is there a way to rescue an antibody solution after the milk has gone
sour?
2) is there a way to preserve the antibody solution any better then
described?

Antibody in question is a Hif 1 alpha from BD, current time until it gets
sour is only 4 weeks or so.
We use 5% dry milk in TBS/Tween.


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  #2  
Old 09-30-2005, 03:37 PM
DK
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Default antibody solution in milk gettin sour fast

In article <433d32e8$0$11352$[Only registered users see links. ].at>, "MMu" <[Only registered users see links. ]> wrote:

I always dilute primary Ab into 3% BSA in TBST containing 4 mM
azide. This way, it can be stored and reused literally for years.
Secondary is always in milk - cleaner and cheaper and secondary
are usually not worth reusing.

DK
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  #3  
Old 10-04-2005, 10:20 AM
MMu
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Default antibody solution in milk gettin sour fast


"DK" <[Only registered users see links. ]> schrieb im Newsbeitrag
news:dhjm4k$1ff$[Only registered users see links. ].wisc.edu...

Thanks, I will try that! Funny that I never thought of using BSA instead...


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  #4  
Old 10-08-2005, 05:32 PM
Dr Engelbert Buxbaum
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Default antibody solution in milk gettin sour fast

DK wrote:



Azide is fine when used with phosphatase coupled antibodies but
inactivates peroxydases. If the latter is used for detection, azide
should be replaced by timerosal (also available under other trade names
like mersalyl).
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  #5  
Old 10-09-2005, 03:04 AM
Apuzzlepiece@gmail.com
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Default antibody solution in milk gettin sour fast

Is there a reason you keep your dilutions for 4 weeks?
I make my TBST fresh every time, keep it for three days, max.

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  #6  
Old 10-14-2005, 06:37 AM
DK
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Default antibody solution in milk gettin sour fast

In article <di8vsb$sbq$01$[Only registered users see links. ]>, Dr Engelbert Buxbaum <[Only registered users see links. ]> wrote:

No need to with primaries. Washes between primary and secondary
remove azide sufficiently (I'd say factor about 10^5) to allow usage
of peroxidase as a secondary reagent.


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  #7  
Old 10-14-2005, 06:39 AM
DK
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Default antibody solution in milk gettin sour fast

In article <1128827041.842135.128450@g14g2000cwa.googlegroups .com>, [Only registered users see links. ] wrote:

I don't know about OP but sometimes you get 10 ul of primary
Ab from someone and there is whole lot of blots to do
and one also always wants to have 50% backup in case something
else comes up later.


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  #8  
Old 10-14-2005, 11:26 AM
MMu
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Default antibody solution in milk gettin sour fast


<[Only registered users see links. ]> schrieb im Newsbeitrag
news:1128827041.842135.128450@g14g2000cwa.googlegr oups.com...

We try to get as much use out of them as possible. Most of the antibodies
work for a very long time and a lot of blots before getting faint.

We discards second AB's after one use however (HRP activity getting low and
because of low volume needed - 1,5 uL or less for a blot).



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  #9  
Old 10-21-2005, 12:27 PM
Dr Engelbert Buxbaum
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Default antibody solution in milk gettin sour fast

DK wrote:



True. But it is much more convenient to use the same solution throughout
the entire procedure, and less error prone too.
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