Hi,
I have been looking at formation of a peptide multimer bound to a streptavidin scaffold. The peptide is biotinylated, so should bind inherently with the streptavidin backbone.
Currently, I have been quantifying concentrations using absorbance values from the unreacted substrates followed by the reacted product after passing through a zeba column to remove small particles, whcih should include any unbound peptide.
The concentrations look OK, and product concentrations are similar to what I would expect, knowing the stock concentration of streptavidin and absorbance produced.
However, when I am applying the construct in downstream cell treatments, the results are quite variable between batches. I wondered if anyone knows of some QA type tests I could run to confirm the multimer is forming correctly?
Thanks in advance for any help :)
Brittany
it is difficult to tell what you are doing from your explanation. Maybe you can explain your assay better. If the peptide is biotinylated and the multimerization product is biotinylated, can you label the peptide with something, like a fluorophore, so that when you perform a spin column, and pull down the product with streptavidin beads perhaps, you can more easily quantify.
Sorry Marcia.
For the assay, I am reacting an eight times excess of biotinylated peptide with streptavidin backbone to produce a targeting construct, without a label. (I am seperately forming this as a FITC labelled construct, with the FITC directly conjugated to the streptavidin but this is not useful for all of my protocols).
For the unlabelled construct, I calculate the stock concentration of substrates (peptide and streptavidin) by absorbance at 280nm using their hypothesised extinction co-efficients, in order to react an eight times molar excess of peptide to streptavidin - which should saturate the four binding sites on streptavidin, even if the biotinylation of peptide is inefficient.
This is reacted at room temperature for 1hr with rotation, and then passed through zeba columns to remove remaining peptide.
I am then calculating the concentration of the tetramer, by taking the initial peptide absorbance *4, assuming each molecule of the tetramer holds four peptide molecules. The final concentration ends up similar to the stock concentration of streptavidin based on these calculations, which is what I expect. However, I am not 100% confident in this method, as the streptavidin produces inconsistent readings on the spectrophotometer with increasing concentrations.
I have been running pull down assays also. I am just looking for ideas to confirm that the construct is successfully forming as i'm quite new to this technique.
Best wishes
Brittany
If you want to know if the complex is forming, between streptavidin and biotinylated peptide, I would label the peptide with a color group or fluorophore, and then use streptavidin magnetic beads to pull down the peptide. Resyn Biosciences sells them. The amount of color or fluorescence you see after you collect and wash the beads, is proportional to how much streptavidin you have. You can also label the peptide with HRP if you want a very sensitive assay. I can tell you how to do this. Or just look up our paper in researchgate on the colorimetric amplification system we used for proteases.
if you need to quantify how much streptavidin you make, then this is a different story. I would label the biotinylated peptide, use it in excess, and then do the spin column to isolate the streptavidin complex. A280 readings aren't very accurate. If you just have streptavidin and don't have other proteins present, then I would use a BPA type protein assay to quantify how much protein you have. This won't work if it is a complex biological mixture.
For quantifying streptavidin, you can always do an ELISA.
Brittany: I'd suggest to use dot blot similar to Western blot by simply using commercially available streptavidin-HRP to detect increasing concentrations of your conjugated biotin-peptide spot on PVDF membrane. Second, analyze the purity of biotin-peptide by HPLC to be sure of a single peak. With solid data on both assays, you are good to do the next experiment.
If you have access to it, native protein mass spectrometry should allow you to determine whether you have made the peptide(4)-streptavidin product.
How are you determining the concentration of your biotinylated peptide? I am assuming it was chemically synthesized with the biotin and then HPLC purified, but the purity would depend on what was specified (higher purity means higher cost and lower yield). The weight of a dried peptide cannot be used to determine concentration since the dry weight includes salts that can be up to 40% of the dry weight, and then if the peptide is only 80% pure your net biotinylated peptide could be less than 40% on a per weight basis.
Brittany Wingham Hi Brittany, I am doing something similar. My peptides are just 20 aa with one cysteine in the N-terminal. I want to conjugate them to Streptavidin beads. Could you please share if you were able to determine the concentration of your conjugates and how you did so? Also, could you please tell your initial reaction volume and concentrations? Thanks.
Hi Brittany, I'm doing something very similar to what you described back in 2015. I've been tring biotin-4-fluorescein to confirm/quantify binding of my biotinylated peptide to streptavidin. It's not working great. Have you identified a better way of doing this?
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