gel percentage & loading control for ATM and ATR? - western blotting (Mar/03/2008 )
what woulod be an appropriate loading control for ATM and ATR? and also what is the suitable SDS-PAGE percentage?
I always analyse proteins ranging from 15 kDa to 115 kDa using 10% and 12.5% minigels, but this seems to be inapplicable for ATM and ATR proteins since their sizes are about 250 and 350 kDa.
any cooments are welcome
Hi, in my opinion, for theese kind of proteins you may need a gradient gel, for example 4-20% or 6-16%. Invitrogen has many pre-casted gel, you can take a look.
yep, Probably your best bet is the Bis-Tris 3-8% gel from Invitrogen- It works well for my large MW proteins and transfers for blots well.
If you can't get these easily, then try a 7% SDS gel or hunt for a gradient gel recipe, you maybe able to make one yourself- though the bought will be much easier and more reliable.
As to a suitable loading control I'm not sure what you mean- surely you need a positive tissue or protein sample containing these proteins, its easy enough to find a high molecular weight marker for these gels- BioRad and Invitrogen certainly have choices.
Lost in the Lab
is their a recipe for gradient gel that I can prepare by myself? if so could you please tell me about it. also in this case would alpha tubulin be a suitable loading control?
you can use your own recipe (you can add different amounts of sucrose to the solutions to strengthen the gradient). just remember to reduce aps and temed to slow polymerization (you wouldn't want the solutions to polymerize in the gradient former).
how you set up the mixer and pour depends on the apparatus, whether you pour from top or through the bottom, size of the gel, how many you are pouring, etc.
I'm afraid I've never poured my own gradient gel, so I have no protocol. however, you should be able to use alpha-tubulin as a loading control if you use a gradient gel.
i'm trying to transfer ATR too but i'm not able to...
someone could provide me a detailed protocol??
which transfer method you use which buffer for how long??
thank you so much