Hi all, I am doing cell surface density measurements of different membrane receptors.
I am using anti HA antibodies against the tagged receptors. Then using Amplex Red method to do the measurements.
I got some problems and I am looking for another method.
Any one has got an Idea?

Hi,
I have got alot of Ideas,
1- try blocking with 5% bovine serum for 30 min on Ice prior to incubation with primary antibody.
2- Wash with PBS + 1mMMgCl + o.1mMCaCl at least 4 times after eah incubation.
If got more problems let me know
Guy

GFP tag your proteins and use InSpeck green fluorescent beads (molcular probes) to standardize your GFP fluorescence to quantify your surface expression.

Example papers of membrane protein quatification using this method can be found at

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12451126&query_hl=1&itool=pubmed_docsum

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11166366&query_hl=1&itool=pubmed_docsum

Thanks for links.
I am using an HA attached protein, so the GST isnt helpfull for now.
Guy

Hey man, I just found an article which might answer some of your problems---------------
Faraday Discuss. 1998;(111):289-305; discussion 331-43. Related Articles, Links

Analysis of membrane protein cluster densities and sizes in situ by image correlation spectroscopy.

Petersen NO, Brown C, Kaminski A, Rocheleau J, Srivastava M, Wiseman PW.

Department of Chemistry, University of Western Ontario, London, Canada.

Communication between cells invariably involves interactions of a signalling molecule with a receptor at the surface of the cell. Typically, the receptor is imbedded in the membrane and it is hypothesized that the binding of the signalling molecule causes a change in the state of aggregation of the receptor which, in turn, initiates a biochemical signal within the cell. Subsequently, many of the occupied receptors bind to membrane-associated structures, called coated pits, which invaginate and pinch off to form coated vesicles, thereby removing the receptors from the cell surface. The state of aggregation of membrane receptors is obviously in constant flux. Any useful approach to measuring the state of aggregation must, therefore, allow for dynamic measurements in living cells. It is possible to use fluorescently labelled signalling molecules or antibodies directed at the receptor of interest to visualize the receptor on the cell surface with a fluorescence microscope. By employing a laser confocal microscope, high resolution images can be produced in which the fluorescence intensity is quantitatively imaged as a function of position across the surface of the cell. Calculations of autocorrelation functions of these images provide direct and accurate measures of the density of fluorescent particles on the surface. Combined with the average intensity in the image, which reflects the total average number of molecules, it is possible to estimate the degree of aggregation of the receptor molecules. We refer to this analysis as image correlation spectroscopy (ICS). We show how ICS can be used to measure the density of several receptors on a variety of cells and how it can be used to measure the density of coated pits and the number of molecules per coated pit. We also show how the technique can be used to monitor fusion of virus particles to cell membranes. Further, we illustrate that, by calculating cross-correlation functions between pairs of images, we can extend the analysis to measurements of the distributions as a function of time, on the second timescale, as well as to measurements of the movement of the receptor aggregates on the surface. Finally, we illustrate that, by this approach, we can measure the extent of interaction between two different receptors as a function of time. This represents the most quantitative measurement of the extent of co-localization of receptors available and is independent of the spatial resolution of the confocal microscope. The theory of ICS and image cross-correlation spectroscopy (ICCS), focussing on the interpretation of the data in terms of the biological phenomenon being probed, is discussed.null

Thanks for the Corrections,
As you said lots of cloning to do.
I will think about it.
Guy