High MW proteins are much slower to transfer. Most probably, in your case
you are not transferring any at all. Try longer transfer times. Some people
posted here before that supplementing the transfer buffer (Tris-Gly-20%MeOH)
with 0.05-0.1% SDS helps, too. Also, check if you are properly inhibiting
proteases in your lysates. Adding peptide inhibitors, EDTA and PMSF would
"tiger" <[Only registered users see links. ].hk> wrote in message
news:bn8phr$nq2$[Only registered users see links. ].cuhk.edu.hk...
hi, anyone: I want to ask some questions of GFP fusion protein western blot
I have constructed a vector of GFP fusion protein and transfected into
HEK293 cells using lipofectin. I check the protein expression under
Fluorescence microscope 48hr after transfection, it is about 60% cells give
green fluorescence in the GFP control cells and 35% cells in my GFP fusion
protein tansfected cells(localized in nucleus, it is nuclear protein). It is
overexpressed. when I did western blot, I can detect the band of GFP in
control (it is not strong). But I can't find the band of my fusion protein.
I lyzed the cell with RIPA buffer, loaded 10ug of whole cell lysis,
transfered to PVDF membrane. first antibody ( anti-GFP ployclonely
antibody1:1000, santa cruz) and secondary antibody (1:10,000 amersham), ECL
(amersham). the molecular weight of protein is about 120kd(with the GFP
I think the proportion of my protein may be too low in cell lysis to detect
it, maybe I should load more cell lysis. Or I should do the nuclear extract
of transfected cells. Or I should change antibody.
All suggestions are appreciated. thanks in advance!
I'm having similar problems with plant material, I assume with control
you have free GFP?! Anyway, fusion protein generally lot less, and
additionally some mutants break down more easily, resulting in more
Have you tried antibody for the protein itself?
"EK" <[Only registered users see links. ]> wrote in message news:<r7Tlb.27$[Only registered users see links. ]>...
1. I can see the fluoresecence of my fusion protein, it is
expressed. -------- the fusion protein expression is ok
2. I can detected the expression of GFP protein (vector only) in the western
blot--------- the detection system should be ok
so I think the problem should be in the sample preparation or protein
As my fusion protein is nuclear protein, I don't know whether the RIPA(1x
PBS,1% Nonidet P-40 ,0.5% sodium deoxycholate, 0.1% SDS, 1mM PMSF and
Protein inhibitor mixture) can completely lyse the cell (especially the
nuclear protein). At same time even it is lysed, the proportion of my
protein in the whole cell lysis may be low. Maybe I should try the nuclear
As the GFP is about 27kd, it is easy to transfer. I don't know whether my
protein(120kd) can be properly transferred. Now I used the three-buffer
system with constant current 60mA for 1.5 hr(Anode buffer I Tris0.3M PH
10.4, Methanol 10%; Anode buffer II Tris 25mM PH 10.4,Methanol 10%; Cathode
buffer Tris 25mM PH 9.4, Glycine 40mM, Methanol 10%). there is still some
bands in gel after transfer. I don't know whether the transfer is ok or not,
maybe I should try longer transfer time.
I didn't have the antibody of my protein. The anti-GFP antibody is
multiclonal antibody (Santa Cruz), it should be ok (I can detect the GFP
protein, even there are some nonspecific bands). Maybe I will also try
antibody of other companies last.
Thanks for all your kindly helps!
"EK" <[Only registered users see links. ]> wrote in message
news:r7Tlb.27$[Only registered users see links. ]...
Sometimes it happens that an antibody does not recognize a modified protein
anymore. Do you have some data on where the antiGFP binds? You might add
some other epitope like vsv angainst which you may obtain monoclonals.
make sure your transfer is complete. Try a longer transfer time (e.g
double, but don't increase voltage or current). Stack two membranes if
you're afraid of loosing your protein. Put another membrane on the *other*
side of the gel if you're paranoid. GFP has a strange structure (barrel)
and nuclear proteins might have strange charge distribution (some nuclear
localization signals a highly posively charged stretch of Arg and Lys).
Try another transfer method (use eg liquid transport without electricity
and use one membrane on each side of the gel)
To get an idea about sensitivity of your antibody, you might probe a dot
blot with various dilutions of your protein (or cell extract). Sometimes
this works: Hold a vial with your extract into the blue beam (490 nm) of a
photometer, mercury or xenon lamp or a 488nm laser if available (confocal
microscope, FACS, ...) to guess some green fluorescence. If you have access
to a fluorescence imager, test your gel and membranes for green bands.
>In article <bnbgir$2d93$[Only registered users see links. ].cuhk.edu.hk>,
Sean Patterson, Ph.D.
CONICET Associate Scientist
Department of Morphophysiology
School of Medical Sciences
Cuyo National University
Tel: (0261) 449-4001 ext.2684/2747
Fax: (0261) 449-4117
e-mail: [Only registered users see links. ]