At a particular conc. I am getting different values of uptake rate in two different experiments. One is uptake experiment where 'X' is used in 10uM conc. Another is saturation kinetics from 5-50uM conc which includes 'X' as a conc too. Is it ideal to get same or similar values of uptake rate at 10uM conc in both the experiments? Everything in the experiment remains same i.e. time of exposure, conc of label and protocol.
Say I get a rate of 62 pmol/mg protein/ min in uptake exp and 32 pmol/mgprotein/min in kinetics. Although the kinetics follows non linear pattern and I get a desired Km value, my data points differ. What is possibly wrong here? All datasets are validated over n>2. Please help.
Hi. I'm confused. When you say uptake experiment, do you mean cellular endocytosis assay?
Hi,
Uptake experiment is intracellular uptake or you could say transport. I provide 'X' , labeled and unlabeled in a ratio in the extracellular medium and assay how much is being taken in as a measure of tritiated radio ligand in the intracellular fraction or lysate after washing the external medium many times. Readings of the cell lysate are taken in scintillation counter. The treatment is in a culture plate/ well in glioma cells. Basic idea is to assess the transport ability of the surface expressed receptors in control Vs mutants.
The way you describe the uptake experiment indiates to me that you are not trypsinizing the cell layer to determine endocytosed ligand. If you lyse the cells without removing cell surface proteins then you are also counting cell surface bound ligand as internalized.
I know from working with LDL receptor family members that their ligands can adhere to many extracellular binding sites besides the receptors.
Are you using a natural ligand for the receptor?
Hi,
Yes I am using a natural ligand and the cell surface expression from previous literature suggests that there are no non specific binding sites for this. Plus the expression of other receptors which could bind to this are not expressed in this cell line, hence this glioma is chosen based on specificity. The empty vector control and the stop codon generated for the protein give basal readings as the untransfected cell population.
Ah. So you are expressing a non-endogenous receptor in this cell line. That could complicate things. Do you know if the cell line expresses the required adaptor proteins for this receptor to endocytose? Does the receptor recycle?
I think that first you need to verify that this receptor is truly internalizing the ligand. The easiest way to verify this is to trypsin treat the cells. If the ligand is internalized then it should be trypsin insensitive and in the cell pellet.
Hi,
Its a standard assay in the field with loads of publication. Yes it expresses endogenously and its a well know transporter molecule. I am overexpressing hence i have a empty vector control. And I subtract the basal level count from untransfected (endogenous) cells. My only worry is, do variability in two different kind of experiment at a same conc. point of view valid or is it unusual in the field. Can I match a conc point from a MM kinetics graph to a single conc transport experiment. Thats my worry.
Using the same conc. of ligand should give you the same level of endocytosis for the same cells IF; 1) they have the same number of receptors, 2) all receptors are functional, 3) you are using saturating conc. of ligand, 4) you can distinguish cell surface bound from internalized ligand.
Is the ligand degraded? That could be another way to determine receptor functionality.
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