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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Loading Control - Western Blot Forum

Loading Control - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 07-09-2007, 05:47 PM
Pipette Filler
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Default Loading Control



I was wondering if someone had advice on using beta actin as a loading control. I've just been reintroduced into the world of molecular biology after over 5 years and I don't clearly recall how you use a loading control. What would you recommend diluting the antibody in and at what concentration? The product sheet recommends 1:1000-1:10,000. Would the beta actin antibody go into your sample and how much would you use? Is this correct? Thanks.
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Old 07-10-2007, 01:07 PM
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Default Re: Loading Control

Hello montMolec!
welcome to the forums. I have heard recently that even beta-actin is not the best control. Many people use alpha-tubulin. What I would do is ask your lab mates or read up on similar papers using the same treatment conditions as controls tend to vary depending on what you are doing to your cells.

Also, antibody manufacturer's want you to use up all your antibody.
Try 1:10,000 that should be enough.
If you want, cleverly run a gel, and transfer to a western blot membrane. Cut the membrane into 3 pieces. Try 1:1000 antibody dilution with one, 1:10,000 antibody dilution with the other, and a different blocking technique with the last piece (1:10,000) ie block also during antibody incubation step.
See what you find

No the beta-actin is present in all your samples, and is usually a standard control (or loading control). You can strip your membrane and blot for this after your initially screen for your protein of interest using your antibody.

Any questions or suggestions please post back here montMolec!
cheers!
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  #3  
Old 07-10-2007, 03:38 PM
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Default Re: Loading Control

assuming we are talking mammalian here, i use gapdh as internal control on my westerns, works nice, but alpha actinin and beta tubulin are also nice. if you are detecting proteins that differ in size i think it is better to cut the membrane and probe each piece with the corresponding antibody rather that strip, you always gonna loose signal.

regarding the dilution, md suggestion is right, try different ones, althoug as a rule of thumb seeing that beta actin is quite abundant you prob would get away with 1:500 to 1:10000. i would dilute it in the same buffer i use for blocking (milk, gelatin, bsa). personally i do my westerns in 5% milk in either pbs-t or tbs-t (t= tween 20 @ 0.1%) because it is cheap,although some people claim is dirtier and harsher that for example gelatin. how much you would load depends in the protein you are probing for, but 20-50 ug total lysate could be a start if your protein of interest if not very scarce...
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Old 07-12-2007, 11:58 AM
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Default Re: Loading Control

Thank you for the welcome and the advice. I will give your suggestions a try and let you know how it works out.
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Old 10-21-2007, 05:49 PM
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Default Re: Loading Control

hello,
i had a simple question about how you even do the internal control. say i wanted to use B-actin (even though i know some of you are saying tubulin is better), do i add the B-actin antibody in the same solution as i add my primary antibody and shake them both at the same time? thanks.
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Old 11-20-2010, 02:14 AM
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Default Re: Loading Control

Yes I am interested in how you actually apply a loading control (eg b-actin) and a 'protein of interest' primary antibodies. Do you mix them together and co-incubate at 4C overnight? Or if you want to re-use the antibody can you 1) block, wash with TBST, apply b-actin in blocking buffer for 1 hr at room temp then 2) wash in TBST, re-block, wash in TBST, then apply second primary antibody for 1hr at room temp. Cant find a protocol online for this but a loading control is normal for published papers. Please help !!! Thank you !
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