General Protocols

General Protocols:

Site directed mutagenesis

Calcein staining of macrophages

Cytokine ELISA 

  • This is an updated version of the original.

How to titrate antibodies for DIY ELISAs

  • It’s often cheaper to buy the components of an ELISA (i.e. standard, capture & detecting antibodies) to make your own ELISA than to buy commercial ones. Here is an easy to follow protocol on how to systematically determine if you are using the right concentrations of antibodies.


  • This is a cheap and effective protocol for transfecting HEK293T cells using PEI.  Note: In Jan 2013 we posted an updated version (version 2) with clarifications requested by users. We have adapted this protocol for 24 well plates. To see the adapted version click Protocol for 24-Well Plates

Western immunoblotting

Macrophage Killing Assay

  • How do you measure how well macrophages kill bacteria (or whether your treatment alters killing)? This is a standard kill curve protocol and a workhorse of the Bowdish lab. Specifically written to measure S. pneumoniae killing, can be adapted for any bacteria and any type of macrophage (e.g. human, murine, alveolar, bone marrow,etc).

Methylene Green SDS Detection Assay

  •  Is there SDS contamination in your reagent? Quantitate the amount of SDS using this assay based off of the traditional Methylene Blue assay.

Protein A Sepharose purification of Antibodies

  • This protocol is useful for purifying antibodies from hybridoma cultures and serum.

How to do a journal club.

  • This “how to” was put together by Prof. Mark McDermott and is designed to help anyone present a manuscript at journal club. The paper he deconstructs is: Evidence for a Common Mucosal Immunologic System Migration of B Immunoblasts into Intestinal, Respiratory and Genital Tissues J. Immunol. 122:1892 (1979)