(by pjie2 from cam.ac.uk)
Sat Sep 25 14:19:51 EST 2010
On 25/09/2010 18:15, DK wrote:
> Peter, sorry to be skeptical but - Occam's razor - I find it hard to believe.
> More details perhaps? How do you detect expression of this protein?
> I hope by Western using some really specific primaries. No signal
> at all? Not even against GFP?
In terms of AB to my protein, we have a polyclonal peptide antibody
raised to a stretch near the C terminus. It detects a testis-specific
band of the expected size (~55kDa) by Western blot. There are two
weaker higher bands (~75kDa and ~110 kDa) which appear to be
non-specific since they're seen in multiple tissue types - the mRNA for
the gene is testis specific. Affinity purified AB shows the same three
bands as the unpurified AB.
Western blot of a developmental timecourse in testis shows that the
50kDa band follows the same pattern as the message for the gene of
interest: first appearing at 19 days post partum, strengthening through
to adult testis, concurrent with the appearance of spermatids in the
testis. IHC shows a signal in the same cell types (round and elongating
spermatids) as the mRNA message.
However, we can't compete the Western signal or the IHC signal with the
cognate peptide. It's thus possible that the AB is detecting a
non-specific band that just happens to be of the right size, in the
right cell type. On the other hand, the fact that the affinity purified
AB shows the same bands suggests otherwise. As to why we can't compete
the signal, maybe the affinity for the protein is higher than the
affinity for the peptide? That's one of the reasons for trying to
express the protein in culture - to help demonstrate that the 55 kDa
band is the right thing!
As for the culture systems we've tried, here's the rundown. All
constructs were of course sequenced fully to confirm the insert was
in-frame with the tag and unmutated.
* pEXP5-CT vector (T7 promoter, C terminal His tag):
Anti His antibody detects nothing at the correct size, although cranking
up the exposure / amount does eventually give a background smear,
especially in the insoluble fraction. Peptide AB for my protein detects
nothing. In vitro transcription/translation using a bacterial kit (E.
coli lysate) shows nothing with anti-His AB or with the peptide AB. In
vitro coupled transcription/translation using a mammalian kit
(reticulocyte lysate) shows nothing with anti-His, nothing with the
peptide AB, and no incorporation of biotinylated lysine - i.e no
translation of anything at all.
Positive controls for the bacterial cultures: three other His-tagged
proteins expressed fine and gave good signal at the right size both with
anti-His AB and with appropriate specific ABs.
Positive controls for E. coli in vitro system: other His-tagged proteins
Positive controls for reticulocyte lysate system: luciferase control
from manufacturer gives functional luciferase, and also shows
incorporation of the biotinylated lysine.
* pT7CFE1 vector (T7 promoter, C terminal His tag):
Same results as the pEXP5-CT vector in both E. coli and mammalian
coupled transcription/translation systems, except that in addition we
isolated the mRNA and showed that the construct was being expressed but
* pGEX(KG) vector (tac promoter, N terminal GST tag):
Peptide AB detects nothing. AB to GST detects a band around the same
size as native GST. The GST band for the fusion construct was
fractionally larger than the band in the empty vector control, so it's
possible we were getting GST plus the first couple of aa of my protein.
Positive controls: Two other GST-tagged proteins expressed fine, at the
right size. Empty vector gave a strong band with anti-GST at the right
size for native GST.
* pEGFP-N1 vector (CMV promoter, C terminal GFP fusion):
There was a low diffuse GFP signal in the transfected cells (HEK293 /
HEK293T). Western shows no band with AB to my protein, anti-GFP shows a
band approximately the size of native GFP and no other bands. In this
case, since it's a C terminal fusion, the band at native GFP size can't
be due to premature termination - we think it's either internal ribosome
entry or just low level contamination with uninserted vector. There's
no evidence of the latter when sequencing the plasmid prep though.
Positive controls: Four other GFP-tagged proteins expressed fine and
gave fusion proteins of the expected size. Empty vector gave a strong
band with anti-GFP at the right size for native GFP.
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