Cell surface biotinylation assays: Cells were washed three times in ice-cold HBSS and then exposed to 0.5 mg/ml Sulfo-NHS-SS-biotin (Pierce) for 30 minutes at 4 °C. To quench excess Sulfo-NHS-SS-biotin, cells were washed three times in HBSS with 5 mM Tris (pH 7.4). Following this step, the procedure was adapted to the goal of the experiment. (1) For determination of cell surface receptors, cells were lyzed in RIPA buffer and centrifuged for 20 min at 20.000 x g. Clear supernatants were incubated with EZview Red Streptavidin Affinity Gel (Sigma) for 4 hrs at 4°C. Beads were sedimented by centrifugation at 1000 g for 5 min, and washed four times with RIPA buffer. Aliquots of input and precipitate samples were analyzed by Western blot using appropriate antibodies. Chemiluminescent Western Blot signals were quantified by a ChemiDoc XRS luminescence imager (BioRad) in combination with Quantity One software. (2) for endocytosis: Cells were stimulated with agonist in prewarmed (37 °C) DMEM growth media for the appropriate time at 37 °C. Remaining biotin label from cell surface proteins was removed by washing the cells twice in ice-cold glutathione stripping solution (50 mM glutathione, 75 mM NaCl, 1 mM EDTA, 10 % FBS, 75 mM NaOH; 2 x for 30 min at 4 °C). To remove residual glutathione solution cells were washed twice in ice cold HBSS. Cell lysis and quantitation of biotin-labeling was performed as described in 1. (3) for degradation: Cells were stimulated with agonist as above. Remaining biotin label from cell surface proteins was removed by washing the cells twice in ice-cold glutathione stripping solution (30 min at 4 °C). Glutathione-stripped cells were again incubated with prewarmed growth media for 3 h or 6 h in a CO2 incubator at 37 °C, followed by cell lysis and quantitation of biotin-labeling. Ref: Turvy DN, Blum JS. Biotin labeling and quantitation of cell-surface proteins. Current protocols in immunology / edited by John E Coligan [et al 2001;Chapter 18:Unit 18 17

Hello Sven,

I can try to help you concerning the biotinylation of your protein. There is a biotinylation system available from Avidity (www.avidity.com) that works in E.coli. It consists of a plasmid (pAN or pAC) which contains a tag (it's called AviTag). You can ligate your gene directly after the AviTag. Afterwards you insert the plasmid containing the AviTag+gene in E.coli cells, that already have a second plasmid carrying the gene of the biotinylation enzyme BirA (pBirAcm). Both plasmids are IPTG inducible, so your protein is expressed with the AviTag and also the enzyme is expressed. The enzyme recognizes the AviTag and adds a biotin to it (to a Lysine residue). And that's it. You have your protein biotinylated inside the cells. Afterwards you can purify the biotinylated protein as usual.

Hope this helps with your first question,

Good luck,

Alex

Hello Alex,

this is very interesting, but I have another problem (but maybe your method is the only right one!). I want to biotinylate cells in a cell culture dish. No E. coli, I have endothelial cells and also carcinoma cells. So I have no gene at the moment which I can ligate after a tag :-(

Then have a look at this link.

http://www.piercenet.com/browse.cfm?fldID=62480CE7-7A66-45E6-B123-D09F4E0F9371&gclid=CMykj-HN4rMCFVG6zAodAHoAwA

Maybe you find something that will help you.

good luck

Hi Sven,

I dont think it is possible to label(biotin/HIs tag/N15 etc..) any individual protein after its expression in the cell (E.coli/human cells). If you want to use a tag sysemtem it is always suggested to go for heterologous expression. May be you can make a stable cell line with a viral vecors?

Hello Ananda,

why did you think this is not possible? Will be very intresting to know.

Cell surface biotinylation assays: Cells were washed three times in ice-cold HBSS and then exposed to 0.5 mg/ml Sulfo-NHS-SS-biotin (Pierce) for 30 minutes at 4 °C. To quench excess Sulfo-NHS-SS-biotin, cells were washed three times in HBSS with 5 mM Tris (pH 7.4). Following this step, the procedure was adapted to the goal of the experiment. (1) For determination of cell surface receptors, cells were lyzed in RIPA buffer and centrifuged for 20 min at 20.000 x g. Clear supernatants were incubated with EZview Red Streptavidin Affinity Gel (Sigma) for 4 hrs at 4°C. Beads were sedimented by centrifugation at 1000 g for 5 min, and washed four times with RIPA buffer. Aliquots of input and precipitate samples were analyzed by Western blot using appropriate antibodies. Chemiluminescent Western Blot signals were quantified by a ChemiDoc XRS luminescence imager (BioRad) in combination with Quantity One software. (2) for endocytosis: Cells were stimulated with agonist in prewarmed (37 °C) DMEM growth media for the appropriate time at 37 °C. Remaining biotin label from cell surface proteins was removed by washing the cells twice in ice-cold glutathione stripping solution (50 mM glutathione, 75 mM NaCl, 1 mM EDTA, 10 % FBS, 75 mM NaOH; 2 x for 30 min at 4 °C). To remove residual glutathione solution cells were washed twice in ice cold HBSS. Cell lysis and quantitation of biotin-labeling was performed as described in 1. (3) for degradation: Cells were stimulated with agonist as above. Remaining biotin label from cell surface proteins was removed by washing the cells twice in ice-cold glutathione stripping solution (30 min at 4 °C). Glutathione-stripped cells were again incubated with prewarmed growth media for 3 h or 6 h in a CO2 incubator at 37 °C, followed by cell lysis and quantitation of biotin-labeling. Ref: Turvy DN, Blum JS. Biotin labeling and quantitation of cell-surface proteins. Current protocols in immunology / edited by John E Coligan [et al 2001;Chapter 18:Unit 18 17

Hello Hans-Jürgen,

thanks you very much for your protocol! Very nice!

Hello again Hans-Jüren,

I just write a private message to you.

Hi Sven, it's unclear if you are trying to assay how long this protein stays on the surface or its overall rate of turnover (half-life). The biotinylation assay would work great if you were looking to measure protein half-life at the cell surface. If you want to determine the protein half-life then a pulse-chase assay would be the gold standard.

Hello Barrington,

thanks for your comment. My idea was to label the cells with Biotin and then because of steric hindrance the specific antibody for my protein of interest can't bind. So after biotinylation the fluoreszenz signal in FACS should be zero. Then after a time of minutes or hours the new synthesized protein occur on the surface of the cell and I can make a staining of this and so I can measure how far the turn-over is. What did you think, is it possible in this way?

Sven, I see now that you are interested in the surface half-life of the protein.

What you are proposing to do doesn't directly answer your question though. What you will get from that experiment is the rate of introduction of new protein onto the surface which you will correlate with loss of the old biotinylated protein. You may run into some problems there. First you are assuming that old protein has to be removed before new protein is introduced.

You are also assuming that as soon as the old surface protein is extricate from the membrane that a new protein replaces it immediately and that the rate of synthesis of the new protein isn't a limiting factor. Even though you said the protein is continually being made the fact is lots of proteins have some basal expression level but require external stimuli to reach functional or some critical level.

Why not fluorescently label your protein on the cell surface (using a specific antibody) and FACS sort over time to determine how quickly you lose surface fluorescence?

Alternatively (and more difficult) you could pulse-label your cells with 35S-Cysteine/methionine, lyse cells, collect and solubilize the membrane fractions (over a few hours), immunoprecipitate your protein of interest, run on a gel and expose to film or a phosphoscreen. You could then easily quantify the rate of turnover of your surface protein.

be careful with antibody based labeling in this type of assay, because in some cases when you use antibodies that label surface receptors it may dimerize or internalize the receptor or mask the binding site with its cognate ligand. If the antibody is validated not to cause these the FACs approach is valid. I have published several papers looking at protein surface (biotoin) and total half-lives (s35). S35 only really works if your protein is being synthesized at a fairly high rate.