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Lab Notebook

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.

Transfection 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.

Electrophoretic 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.

Nuclear Extract Preparation

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 (PDF)

           Chemically Competent DH5a (PDF)

            Transfection of HEK293 cells with Lipofectamine 2000 (PDF)

        SDS-PAGE gel recipes (PDF)

        Repurification of commercial LPS (PDF)
           
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 transfections

        Quantitative RT-PCR (MSWord doc)   How we routinely do it.
                    Also a good reference from Applied Biosystems on qRT-PCR

 

 


         

 

This page last updated: 02/17/04