Protein labeling, immunofluorescence

[Ivaylo]

Hello !! I want to know the machanism of the protein labeling with antibodies. I want to label a protein that is being synthesized in the cell. I don't understand, how this thing is happening... How the antibodies pass the selective membrane?? Thank You.

[CharonY]

You mean you want to visualize a protein in vivo? Usually that is done by marker fusion (e.g. GFP) of the protein in question. Other more error prone protocols involve the uptake of the ABs by endocytosis (in case of plants protoplasts are used). Overall specificity and efficiency can be worriesome, though.

[Ivaylo]

CharonY, thanks for your reply. I found that mild detergents are also being used to make channels in the membrane so the ABs can pass through it.

[CharonY]

Why, yes of course. Overall, I only have limited experience with in vivo antibody labelling (did it only once or twice with plant protoplasts, which took up the conjugates). The rest were done in vitro, that is after fixing the tissue. Do the cells survive the detergent reaction?

[Ivaylo]

I haven't done it, I am making a project and I am facing real difficulties combining the different techniques, that's my first project though and I am quite

inexperienced. Concerning the mild detergent treatment, I think it works cause I found it on the internet, but donno what detergent is being used, for how long and so on, but I think it' s working. CharonY, please check my new topic, I am trying to induce UV mutations in a gene, but the whole thing is one big mess.

[lucaspa]

CharonY, thanks for your reply. I found that mild detergents are also being used to make channels in the membrane so the ABs can pass through it.

 

And the cells are usually dead when this happens. Immunohistochemistry is done either for surface proteins, in which case the cell can remain living, or for intracellular proteins. If intracellular, the cell is dead either by freezing or fixing and means are used to poke holes in the cell membrane.

 

Detergents are one method. In my lab, we fix in methanol, remove the methanol, and then dry the cells. This results in fractures in the cell membrane thru which the antibodies can move.

[lucaspa]

I haven't done it, I am making a project and I am facing real difficulties combining the different techniques, that's my first project though and I am quite

inexperienced. Concerning the mild detergent treatment, I think it works cause I found it on the internet, but donno what detergent is being used, for how long and so on, but I think it' s working. CharonY, please check my new topic, I am trying to induce UV mutations in a gene, but the whole thing is one big mess.

 

The common detergent is 0.1% Triton X-100. You use antibodies to identify the protein once it is made and do immunohistochemistry on dead cells.