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Below you will find some of the protocols
that we routinely use in the lab. If you have any questions about them feel
free to give us a call or email.
of RAW 264.7 macrophages
We have been using the standard calcium
phosphate transfection technique for many years with good success. Other labs have
successfully used some of the lipid-based transfection reagents, but we have found this
method to be the most reproducible and economical.
Mobility Shift Assays
Otherwise known as EMSA or gel shifts,
this assay is used to characterize sequence-specific DNA-binding proteins in crude nuclear
extracts. By incorporating unlabeled competitor DNA or specific antibodies this can
be a very powerful technique for determining the identity of a protein bound to your
particular promoter fragment.
Our modification of the Dignam
et al (Nucleic Acids Research 1983 11:1475) procedure for preparing extracts containing
DNA binding proteins
Histochemical staining of cells
transfected with LacZ constructs
Chemically Competent DH5a
Transfection of HEK293 cells with Lipofectamine 2000 (PDF)
SDS-PAGE gel recipes (PDF)
Repurification of commercial LPS
This is the paper by Hirschfeld, et al. which describes the
additional purification of commercially-prepared LPS in order to remove
contaminating lipopetides (i.e. TLR2-activating stuff). This method
results in E. coli LPS that is a pure TLR4 agonist. We buy our LPS
from Sigma and always treat it this way.
MKN45 and AGS
Quantitative RT-PCR (MSWord
doc) How we routinely do it.
Also a good reference from Applied Biosystems on
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