Native acrylamid gel - (Nov/27/2007 )

Hallo everyone !

Can anyone help me, i need to test my proteins (35 and 50 kDa ) in NATIVE gel and i dont have any protocol or experience

in that field.


Thanks for the help.

hi,

first check for the IP of the protein to know how they will migrate... make appropriate buffer and gels
do not add any SDS nor reducing agent in the loading buffer
do not boil the samples

for the remaining, it is like a normal SDS-gel
but can take longer time for migrate

Sebastien

If you dont have time or experience to optimaize it, you may try precasted 4-20% gradient gel. Several companies sell these.

hi,

first check for the IP of the protein to know how they will migrate... make appropriate buffer and gels
do not add any SDS nor reducing agent in the loading buffer
do not boil the samples

for the remaining, it is like a normal SDS-gel
but can take longer time for migrate

Sebastien

Thats what "native" gel stands for, otherwise its called denatured.

for denaturation you have SDS and BME or DTT BME or DTT to dirsrupt disulfide bridges, and SDS to "linearize" and to get rid of the charge-dependent migration
so, how do you get rid of protein protein interactions? I mean ... how can you see a monomer? it should be a real mess ?

How would using a precast gel remove the "optimization" required to get native gels and EMSAs to work properly...

Misele has the best advice. For a good native gel protocol check out:
Li Q, Cooper JJ, Altwerger GH, Feldkamp MD, Shea MA, Price DH.HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells. Nucleic Acids Res. 2007;35(8):2503-12
http://www.pubmedcentral.nih.gov/articlere...bmedid=17395637

The original post never revealed his/her purpose of study. I interprete that a good separation of the two proteins under a non-denaturing condition is all that needed. For native gels, T% and C% are important parameters for protein separation in addition to the charge differences. For best separation without much trial-and-error, a gradient gel is the best choice. Gels for EMSA (T=~6%) are not the best choice for protein separation. Proteins in that MW range will be poor resolved.

check these threads for native page buffer systems:

high and neutral pH



acid pH

hi and neutral are for most proteins, acid for proteins with high pI or for different selectivity.