Need for help !! Protein reluctant to be electrotransfered in Western Blot

Lena Zaitseva zaitseva at biochem.purdue.edu
Wed Feb 3 10:53:01 EST 1999


=A0=A0=A0=A0 All transfer buffers, which we are working with contain meth=
anol and SDS.
Methanol contents can be changed with time. I would recommend to make a f=
resh
transfer buffer to insure reproducibility.=A0=A0=A0=A0 In our lab we are =
working with
extremely hydrophobic basic protein (theoretical pI is 10.66). This prote=
in is
transferred to the nitrocellulose membrane with very low efficiency in re=
gular
transfer buffer (the protein either stays in the gel, either goes through=
 the
membrane, even through two sheets of membrane). So, increasing of the SDS=

concentration (up to 0.1%) and changing of methanol concentration (10-20%=
) can
improve the transfer. That is good enough for Western. However, for N-ter=
minal
sequence analysis we had to use Immobilon-CD membrane (Millipore) (descri=
bed in
Protein Science (1996), p.1105 - N-terminal sequence analysis).

> > Carlos Duarte wrote in message <01BF6902.37730880 at sida2.cigb.edu.cu>.=
=2E.
> > >
> > >We have been working for several years with a recombinant protein th=
at
> > contains multiple copies of the V3 loop of different HIV-1 isolates.=A0=
 The
> > usual purification procedure in the lab consist of differential washe=
s of
> > the cellular pellet with Urea,=A0 followed by reverse phase HPLC
> > chromatography using a C4 Vydac column.=A0 This protein called TAB9 i=
s about
> > 20 kD and with a theoretical isoeletric point of 11.3.=A0=A0=A0 We ha=
ve studied
> > many times the correct exposition of epitopes in TAB9 with monoclonal=

> > antibodies against V3 by standard western blot procedures using eithe=
r
> > semidry or submarine Biorad transfer apparatus.
> > >However since last month I have been trying unsuccessfully to repeat=
 this
> > procedure.=A0 I load up to 5 ug of TAB9 in 15 % SDS-PAGE and after ru=
nning the
> > sample I attent to transfer the proteins to nitrocelulose membranes
> > (Schleicher and Schuell and Hybond C) However all other proteins seem=
s to
> > transfer fine to the membrane except TAB9 as checked by Poceau red st=
aining.
> > In some experiments we have even developed the technique to the end w=
ith
> > Mabs, but no reactions were observed.=A0 We have tried with recently =
purified
> > TAB9 and with lab standards of this proteins purified 2 years ago wit=
h the
> > same results. We have also tested other labs's reagents and the same =
thing
> > has occurred. I=A0 read that very basic proteins may need a more basi=
c
> > transference buffer and this is the next thing I may try but as I sai=
d
> > before we always used standard transfer buffer=A0=A0 ph 8.3 with good=
 results.
> > >Any suggestions will be very welcome.
> > >
> > >Aristides Aguilar

=A0




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