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antribody gone old?


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11 replies to this topic

#1 maris

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Posted 08 July 2009 - 06:30 AM

Hello,

I have recently done western blotting without success; only faint bands appear. :( Few months ago it worked perfectly. I wonder if my primary antibody has gone old? I received it a year ago and have stored it -80 degrees in small aliquiots. I always use new aliquiot. How long antibody will stay fresh? Is it still possible to use the antibody if I just use it more. Has anyone knowledge about this? Thanks

#2 Gerard

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Posted 08 July 2009 - 07:30 AM

I don't think the antibody is the problem, but maybe you can contact your supplyer if they have information about storageconditions and shell life.
I have several antibodys, primairy and secondary, that have been stored for more than a year at 4oC and are working perfect
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 bob1

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Posted 08 July 2009 - 04:33 PM

Most antibodies will degrade with time, however, this will depend on the storage conditions. Storing at -80 should be fine for several years; even though some antibody data sheets specifically exclude freezing, there are very few that aren't better off frozen for long term storage.

You could try using more of the antibody, but this may not work either. Is it possible that your detection substrate is off, ECL and other detection systems only last for a few months in the fridge.

#4 maris

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Posted 08 July 2009 - 09:03 PM

thanks for replies. I use alkaline phosphatase based detection with nbt/bcip. The expirasy time for the substrate is november 2010 so it should be fine.

I contacted the antibody supplyer and they said that the antibody should be fine. They recommended me to use protease inhibitor during the detection. Has anyone done this? At what stage I should use it and how much?

I wonder if my proteins can degrade during gel run or during transfer. After both runs the buffers are warm. I cannot figure out anything else. After development of 30 min I still cannot see even background. After development overnight I can see background and my protein band but the band is not stonger than the background. I used to get after 5 minutes 1 stong band. I have made newly all buffers. :P

#5 Gerard

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Posted 09 July 2009 - 01:19 AM

Test you detection system, a few drops of your diluted alkaline phosphatase coupled antibody and a few drop nbt/bcip, this should give color in a few minutes.
I'm more worried about you telling over getting warm buffers, could you give more specific details as time, voltage and current.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#6 GeorgeWolff

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Posted 09 July 2009 - 01:59 AM

maris - do you run controls? what were the observations with these?

#7 maris

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Posted 09 July 2009 - 04:16 AM

I just did the activity test for both subtrate solution and secondary antibody. In both tests nice blue color appered immidiately so they should be fine.

I run my gels in room temperature 200 v about 1.5 hours with cold buffer. Today I did the run in cold room and the buffer stayed cold. :P Blotting I perform with 100V for 1.5-2 hours in a cold room with cold buffer. It starts to warm after 1 hour (I dont use SDS). But as I dye the gel after blotting I always see that the bigger proteins were not transferred. Luckily my protein is about 25 kDa and they seems to be transferred. I use PVDF memrane.

What is meant by controls. I have no control samples in my run. Just my protein extracts.

#8 Gerard

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Posted 09 July 2009 - 04:58 AM

The problem seem to be the blotting procedure where the proteins not are transferred quantitative and the big ones sometimes not at all.
Since you didn't have the problem in the past I would check very carefully off something is changed in the procedure and prepare a fresh transferring buffer.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#9 maris

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Posted 09 July 2009 - 05:09 AM

I always prapare fresh transfer buffer day before the transfer and put it into freezer +4. After transfer the buffer is +40 degrees. By blotting 100 V for 2 hours the currency starts at 200 mA and ends with 450 mA.

#10 Gerard

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Posted 09 July 2009 - 05:37 AM

The voltage and current are okay. If the temperature is the problem I'm not sure but I think it's high and possible the proteins degrade.
On the other hand if it's the same as in the past it's not likely.
I've didn't have this problem and I can't think of other possiblities to solve the problem but maybe someones else has.

In my transferunit is a container with melting ice and the buffer is maybe 4 degrees of something like that during the run.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#11 maris

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Posted 09 July 2009 - 06:49 AM

thanks for replies.

Does anyone know sensitive proteins are to heat during transfer? Is 40 degrees allready too much?

#12 Tfal

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Posted 17 July 2009 - 04:48 AM

are you running a Native or a SDS-PAGE? If it's SDS-PAGE, I think you should have SDS in your transfer buffer. Over eating during transfer is problematic. One of the reasons i think is I alters your gel (it kind of cooks it) and some protein might remain stuck in it. I doubt 40 C is problematic. Anyway for SDS-PAGE, you proteins are already denatured. As for a Native, well 40 C is just 3 C above normal body temp, so a vast majority of protein should still have their original conformation.
Are you using PVDF or nitrocellulose? If your are using nitrocellulose and your transfer is too long, your proteins can be pulled through and out of you membrane.

anyway, good luck




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