Western Blot washing

[Oto]

I am trying to get rid of an extra band in my Western Blot. My P.I. recommended I add SDS as an extra detergent to my washes. We use PBST (0.02%) right now. Any ideas on what percentage of SDS to add?

[butters]

extra band? usually that means ur secondary antibody binds to it. may we know what protein/antigen you transfer to ur nc/nylon membrane? and what first and second antibody did you use?

[admin]

I would make sure you add blotto 2-5% milk in your blocking step for 1-2 hours, but also add the milk to your primary and secondary antibody incubations. Will clear up almost all blots.

cheers

[butters]

admin, so far i been blocking with 2-5% percent for 1 hrs seem more than enough. i afraid the second band was other reason beside non specific interaction. but we wait for Oto for more update

[Oto]

Thanks for all of the advice! I actually cleared up the extra bands with longer blocking (10 hrs overnight) with 5% milk in PBST. Also put primary and secondary Ab in 5% milk/PBST. My primary Ab was anti-estrogen receptor and anti-progesterone receptor. Secondary was anti-rabbit. For whatever reason, that worked for me. Although our lab tech tried it for one of his blots and it blocked his band of interest. Whatever works! Thanks to everyone for their suggestions.

[jiajia1987]

hello everyone,

how do you usually decide the %(v/v) of tween to add into PBST? What does tween actually do? all i know is that it is a surfactant!

[jiajia1987]

hello everyone,

how do you usually decide the %(v/v) of tween to add into PBST? What does tween actually do? all i know is that it is a surfactant!

[gulg_genetic]

we use TBST with a ratio of v:v = 1:1000 (tween to TBS), since it is a detergent it helps during the washing steps so you would get rid of unspecific bands..

[jiajia1987]

Thanks for getting back to me.

I use 0.01% Tween-20 in DPBS for my western blot washing.

My supervisor once told me that 0.1% Tween-20 is pretty high.

Thus, I was asking how do we decide how much of the Tween-20 should we have in the washing buffer?

[butters]

I can't really give exact % for tween-20 for wb but usually depended on what the standard protocol in ur place. then adjust from there. The optimum % would probably have to determine experimentally. Not all protein behave the same.