I am trying to Biotinylate the cell surface proteins of mouse neutrophils before pulling them down with Streptavidin-magnetic beads (https://www.thermofisher.com/order/catalog/product/65001#/65001) and then blotting for various GPCRs.
I am using the reducible (as this will be important for future experiments) Z-Link Sulfo-NHS-SS-Biotin (https://www.thermofisher.com/order/catalog/product/21331#/21331) to biotinylate the cell surface and have confirmed this by IF.
I lyse my cells in the following buffer for 3 minutes before a 30 minutes IP with the pre-washed beads.
1% Nonidet p-40 1%
NaCl150mM
Tris pH 7.2 @4°C10mM
EDTA 5mM
MgCl2 1mM
Leupeptin25ug/ml
aprotinin25ug/ml
PMSF0.1uM
Pepstatin25ug/ml
antipain25ug/ml
Beads are boiled for 10 minutes in 1x SDS sample buffer recipe (5% B-ME) and lysates taken forward for western blotting.
As you can see in the images below, I have pulled down some proteins (Coomassie) but there isn't a noticeable difference between +/- biotin. I did not pre-clear the samples in this experiment as I was trying to reduce sample time in the harsh proteases present in cell lysates, but clearly I am getting some stuff sticking to the beads.
The bands detected at ~55kDa and ~30kDa perfectly overlap when present in the 3 blots but do not correspond with the expected sizes of the protein. The blots were stripped between rounds with 10 minute incubation in 1% SDS, 25mM glycine pH2.0 buffer.
In this experiment I did not include a beads only control but in a repeat saw similar bands size at ~55kDa - could be streptavidin?
I have not used these antibodies before and I do not have a positive / negative control for them. I was hoping it to obvious in the +/- biotin samples. I am ordering reagents to overexpress / KO in a cell line.
Any help would be much appreciated
Many thanks
Stephen
One thing to think about is the level of free biotin in the samples when you do the pull-down. This comes from the growth medium and the cells. Streptavidin has higher affinity for free biotin than for conjugated biotin, so if there is any free biotin present, it will act to block binding of the SA beads to the biotinylated surface proteins. The cells should be thoroughly washed to remove all traces of medium before lysis.
Have you tried probing the blot with streptavidin as a positive control? Make sure to use biotin-free blocking solution, not milk, for blocking the blot if you do this.
Hi Adam,
These are primary Neutrophils so I am not sure that free biotin would be an issue here.
I haven't tried blotting with streptavidin yet. In this experiment it wouldn't work well because I am reducing the linker off during my sample preparation, but I think it is a good idea and I will try eluting the proteins and in non-reducing conditions.
Many thanks for the suggestions
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