western blot

[valeria]

I forgot to put the DTT in western blot samples
i can't have another go
the western blot is it destroy or can I use it?
thanks
valeria

[Pow Joshi]

On 18/12/2007, valeria <valeriarix@...> wrote:
>
>
> I forgot to put the DTT in western blot samples
> i can't have another go
> the western blot is it destroy or can I use it?
> thanks
> valeria



you can still interpret the data depending on the locations of your -s-s-
linkages in the protein. DTT or 2-ME are reducing agents that break th -s-s-
bons to form -SH bonds. For eg: if you have antibody sample, the reduced Ab
runs as two bands of ~55kDa and 25kDa while the non-reduced sample will run
at ~150 kDa.

Hope that helps
Pow

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[WS-4]

Valeria,

if you haven't loaded your samples yet on the gel, just add 5 or 10ul
2M DTT, it will work fine. If you already have run the gel, don't
panic (too much), you just did not reduce your samples. They will run
at different sizes maybe. You also might see some addional bands due
to cross-linking and inter-/intramolecular -SS- bridges.

Best,

Wo
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[Han-3]

WS <novalidaddress@...> wrote in news:1479089b-0cc1-4051-aff6-
a2dd19d1b1c7@...:

> Valeria,
>
> if you haven't loaded your samples yet on the gel, just add 5 or 10ul
> 2M DTT, it will work fine. If you already have run the gel, don't
> panic (too much), you just did not reduce your samples. They will run
> at different sizes maybe. You also might see some addional bands due
> to cross-linking and inter-/intramolecular -SS- bridges.
>
> Best,
>
> Wo

In addition, some antibodies don't recognize reduced proteins - they need a
"native", folded protein configuration.

--
Best regards
Han
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