发信人: moso (野鬼), 信区: Biology
标 题: Re: Gel shift问题请教
发信站: Unknown Space - 未名空间 (Mon Apr 11 18:42:02 2005), 转信


yes, the protocol does not mention anything about RNA probe. But it can also
be used for Protein:RNA interation. If you look at the FAQ in the promega's
website, you will find they have some consideration about the probe, like size.
Basicly, if there were binding, EMSA can be applied to detect the interation.
And RNA, single stranded DNA can be 5'end labeled by T4 Polynucleotide Kinase.
After labeling, a chromatographic purfication is used to remove free isotope
so that to reduce the background.
In fact, there were many papers published with EMSA to detect protein:RNA
interactions. Here is one of them,
http://www.qb.fcen.uba.ar/ib
/A%20modified%20quantitative%20EMSA%20and%20its
%20application%20in%20the%20study%20of%20RNA%20protein%20interactions.pdf

Google it, you can find more.

good luck


【 在 batfly (斧头帮帮主玉面飞龙至尊宝) 的大作中提到: 】
: 我查了一下Promega的protocol
: 不过好像只提到了DNA-binding protein?
: RNA的也适用吗？？
: 多谢！！
: 有没有专门讲RNA binding 的protocol啊
: 【 在 moso (野鬼) 的大作中提到: 】
: I think you can go to read some protocols to have a gerneral knowledge before
: starting your work. The Gel shift assay protocol from Promega is a good choice.
: To my experience, EB staining does not work very well expecially to those
: single-stranded probes.
: 【 在 batfly (斧头帮帮主玉面飞龙至尊宝) 的大作中提到: 】
: : 另外对Gel有什么特殊要求吗？
: : 俺对这一块懂的很少，望大侠见谅～
: : 我的蛋白大概有34kD，可能有RNA binding的能力
: : 所以想做gel shift 看一下结合强弱
: : RNA oligo很小，20 bases， 6k 多一点
: : 我看文献大多用同位素标记
: : 但据说比较麻烦，还要过柱分离什么的，
: : 确实如此？
: : 还有就是不用同位素标记
: : 直接做体外结合，
: : 然后用EB染色
: : 不知道哪一种更好?
: : 或者有更方便的方法？
: : 望赐教～


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