Detecting Transcription factors in western blot - Hard time detecting LEF-1(TFs) (Oct/29/2007 )
Hi All,
i am trying for a long time to detect LEF-1 a transcription factor associated with wnt signalling to detect at the protein level. i could very well find the expression levels in RT-PCR but could not get them in WB.
i sonicate the cells in pierce M-PER lysis buffer(it works great) but could not detect these proteins.
i have used all the commercial antibodies but could not detect it. is there any special criteria to make thes TFs stable post lysis because i remember reading a post in this forum that TFs are more labile e.g c-myc. what could be done to circumvent this problem?
i use Roche protease inhibitor cocktail tablets during lysis.
your comments are highly appreciated 
thanks
Rajgene
-rajgene-
QUOTE (rajgene @ Oct 29 2007, 07:39 AM)
Hi All,
i am trying for a long time to detect LEF-1 a transcription factor associated with wnt signalling to detect at the protein level. i could very well find the expression levels in RT-PCR but could not get them in WB.
i sonicate the cells in pierce M-PER lysis buffer(it works great) but could not detect these proteins.
i have used all the commercial antibodies but could not detect it. is there any special criteria to make thes TFs stable post lysis because i remember reading a post in this forum that TFs are more labile e.g c-myc. what could be done to circumvent this problem?
i use Roche protease inhibitor cocktail tablets during lysis.
your comments are highly appreciated
thanks
Rajgene
i am trying for a long time to detect LEF-1 a transcription factor associated with wnt signalling to detect at the protein level. i could very well find the expression levels in RT-PCR but could not get them in WB.
i sonicate the cells in pierce M-PER lysis buffer(it works great) but could not detect these proteins.
i have used all the commercial antibodies but could not detect it. is there any special criteria to make thes TFs stable post lysis because i remember reading a post in this forum that TFs are more labile e.g c-myc. what could be done to circumvent this problem?
i use Roche protease inhibitor cocktail tablets during lysis.
your comments are highly appreciated
thanks
Rajgene
some low expressed proteins are only to detect if much antigen is offered; f.i. in mini gels we use 50 µg per lane to detect p21
-The Bearer-
QUOTE (The Bearer @ Oct 30 2007, 02:21 AM)
QUOTE (rajgene @ Oct 29 2007, 07:39 AM)
Hi All,
i am trying for a long time to detect LEF-1 a transcription factor associated with wnt signalling to detect at the protein level. i could very well find the expression levels in RT-PCR but could not get them in WB.
i sonicate the cells in pierce M-PER lysis buffer(it works great) but could not detect these proteins.
i have used all the commercial antibodies but could not detect it. is there any special criteria to make thes TFs stable post lysis because i remember reading a post in this forum that TFs are more labile e.g c-myc. what could be done to circumvent this problem?
i use Roche protease inhibitor cocktail tablets during lysis.
your comments are highly appreciated
thanks
Rajgene
i am trying for a long time to detect LEF-1 a transcription factor associated with wnt signalling to detect at the protein level. i could very well find the expression levels in RT-PCR but could not get them in WB.
i sonicate the cells in pierce M-PER lysis buffer(it works great) but could not detect these proteins.
i have used all the commercial antibodies but could not detect it. is there any special criteria to make thes TFs stable post lysis because i remember reading a post in this forum that TFs are more labile e.g c-myc. what could be done to circumvent this problem?
i use Roche protease inhibitor cocktail tablets during lysis.
your comments are highly appreciated
thanks
Rajgene
some low expressed proteins are only to detect if much antigen is offered; f.i. in mini gels we use 50 µg per lane to detect p21
ok thtz a good point...i usually load 30µg may be i increase it.
thanks for the comment
-rajgene-