failure with first western blot - (Oct/12/2006 )

Hi,

I just did my first western blot, and not surprisingly, failed. the protein transfer happened successfully, as i can tell from a prestained marker. however, after incubation with secondary antibody, treated with ECL plus reagent, and scanned with Typhoon, the whole membrane is black. (I have attached the figure.) I am wondering if I used too much secondary antibody. the secondary antibody I used is HRP-goat anti-mouse IgG gamma in 1:5000 ratio. 1-hr incubation at room temperature, washed 5 times, 5 min for each.

Can anyone give me some advice? I would really appreciate it!

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Hi,
Don´t worry!!
The same thing happened to me too the first time
Yes, the whole membrane is black possibly because an excess of secondary Ab. After a few minutes, some sites in the membrane (which corresponded to my protein) became white. White bands (negative bands) generally occur when antibody concentration is too high or target protein is in excess. To rectify this, use lower antibody concentrations.
I obtained quite good bands by diluting more my secondary Ab. ECL plus is a quite sensitive substrate. It happened to me using ECL Advance, which is even more sensitive and with this I have to dilute my secondary up to 1:10000 and so I save the expensive Ab.

But your first time is good, neither problem with gels nor with transfer... that´s great.

PD. I think you haven´t attached the figure

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My first time with WB, was a nightmare too. I never got a signal for a month. turns out the antibody wasnt working and it wasnt my fault.

u could use much lower conc. of antibody and also lesser sensitive ECL.

B happy, atleast u have a signal, a bit too much.

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Thanks for your reply. I feel much better now. I will try again!

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Wow, they are really black.
Now I attach my first western and you´ll see that you´re not alone...
Hope this make you feel better. Don´t worry.

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are you sure you blocked the membrane after transfer and before primary antibody?

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what are your blocking conditions, wash buffer, incubation buffers?
also, you might dilute more your secondary antibody.
try increasing dilutions of secondary (without primary) until you don't see background or bands.

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hi
well i did my first blot yesterday and got nothing , even no backround , thts surprising , any suggestions plzz ???

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Could you be more explicit,please? tell us about your protocol, if the transfer was OK...
Could be lots of things

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