Have you been wondering if ordering in bulk is the better move for your upcoming project? Wondering if there is pricing incentive when buying larger quantities? The quick answer is yes!
The best cost per microgram is larger 1 mg size, where we typically give the biggest price break and seeing upwards of a 20% discount when comparing four 250 ug vials and the 1 mg vial.
Another benefit of ordering in bulk is maintaining lot consistency for your project. This allows you to develop a consistent protocol and not have to be concerned with variability in batch-to-batch protein concentrations.
We understand that typically the upfront cost of buying in bulk can be the limiting factor, so I want to take a second and let you know that we also offer plans where you can reserve 1-2 mg over a 12-month period for a specific lot, and just pay for the amount that you draw down.
ATS offers a custom biotinylation service to biotinylate antibodies and peptides, especially when customers plan to use them with our Streptavidin-ZAP.
We have been seeing a trend of variability with commercially available antibodies, where customers will see over or under derivatization of material and this can be an issue if you plan to use it with Streptavidin-ZAP.
There are a few fundamentals to look for when choosing/creating your biotinylated targeting agent:
(1) the linker doesn’t impair target-binding affinity,
(2) the linker should be stable and
(3) the linker should be able to release payload efficiently.
Important details to know about your biotinylated targeting agent:
(1) the ratio of biotin to the protein,
(2) unreacted biotin is purified away
(3) the length of linker because long linkers could interfere with binding and
(4) location of where biotin is attached such that important protein side-chains weren’t impacted.
If any of these parameters are unfamiliar to you or you weren’t given these details, then I strongly encourage you to talk to us about a custom biotinylation service.
Streptavidin-ZAP (streptavidinylated saporin) combines with your biotinylated material to make a targeted toxin.
Unlike a secondary antibody binding to a primary antibody, the bond between streptavidin and biotin is rapid, essentially non-reversible, unaffected by most extremes of pH, organic solvents and denaturing reagents. It is essentially the strongest known noncovalent biological bond between protein and ligand.
Streptavidin-ZAP is super modular and works with biotinylated antibodies, peptides, growth factor, aptamers, anything that will recognize a cell surface receptor and can be biotinylated.
We have kits available which will also include an appropriate control conjugate depending on the species of antibody you’re using or if you’re using a peptide.
There are dozens of publications of using Streptavidin-ZAP in vivo.
When or Why should you switch from using a secondary conjugate (like Fab-ZAP or Streptavidin-ZAP) to a direct saporin conjugate?
If you are working in vitro and using our secondary conjugates to specifically screening numerous targeting agents, then I would say “stay the course” and continue using these types of products. They are quick, effective and economical in screening antibodies.
However, if you are working in vivo and have been using our Streptavidin-ZAP to screen your biotinylated targeting agent, I would strongly suggest you contact us about performing a direct saporin conjugation.
Two reasons why you would want a direct conjugate:
1st is Cost effectiveness. Streptavidin-ZAP is great at assessing your targeting agent, but when looking downstream at the cost to create bulk mg sized batches, a direct conjugate would provide double the yield with equivalent cost.
2nd is Homogeneity. Since the targeting agent is directly-labeled with saporin, we end up with a product that is more homogeneous versus a conjugate made with various components and various labeling.
pHast Conjugates are one of our fastest tools to quantitatively test your primary antibody’s specificity, binding, and internalization, providing results in 1 day.
The pHast conjugate binds to your primary antibody via a secondary antibody cross-linked to a pH-dependent fluorescent reporter. This fluorescent reporter will increase intensity as the pH of its surroundings becomes more acidic, such as you would see on the inside of a cell.
pHast conjugates can be used with any fluorescence visualization device like a fluorescent plate reader, fluorescent microscope and can be used to illuminate your lead antibody candidates with same-day results.
Saporin conjugates can be used to create animal knockout models including Alzheimer’s Disease, Parkinson’s Disease, narcolepsy, epilepsy, and amyotrophic lateral sclerosis (ALS)
Genetic knockouts can be expensive, time-consuming, and with unwanted conditions
Disease models with saporin conjugates are ready in 2 weeks and are less expensive
We have been using your human fab-pHast (PH-01) product for a while now and are very happy with the results in our imaging applications (we have seen signal to noise of over 70k). However I have recently tried running a black bottom plate on a plate reader, same set up, and am getting a high background signal. My signal to noise is about 1.5 in the best case scenario. Could you please advise how to increase the signal to noise in the plate reader format using fab-pHast?
Answer:
We recommend washing the cells with PBS before reading on the plate reader to help reduce background signal. You can find an internalization assay protocol here.
Question:
I’m using complete media (phenol red DMEM with 10% FBS) for my experiments, could this be contributing to background signal?
Answer:
Using phenol red-containing media could contribute to background signal. Replacing the media with PBS should reduce background significantly.
Question:
Do you have any recommendations on whether to use black opaque-bottom plates, black clear-bottom plates, or standard clear TC plates for maximal S/N?
Answer:
Clear-bottom plates offer the advantage of imaging, but they may introduce more background noise. If imaging is a priority, you might need to balance between signal strength and versatility.
Related Products
All of our pHast Conjugates are available with these fluorescent dyes and as a multi-color kit with all four colors in one in one bundle.
The pHast BLUE has an excitation wavelength of 362 nm with an emission maxima at 452 nm. (PH-B)
The pHast GREEN version has an excitation wavelength of 453 nm with an emission maxima at 522 nm. (PH-G)
The pHast RED has an excitation wavelength of 532 nm with an emission maxima at 560 nm. (PH-R)
The pHast FAR-RED has an excitation wavelength of 643 nm with an emission maxima at 660 nm. (PH-F)
Hello, I am very interested in using your Fab-ZAP line. So Fab-ZAPs simply tells us what mAbs internalize best – is that right? Can you send me some example data? I’d love to see a cell line that has been evaluated and the data that has been generated.
Answer:
Yes, Fab-ZAPs will tell you what mAbs internalize best. We have a variety of different versions of these products (ZAP secondary conjugates) which you can find here.
These have been tested on many different cell lines, both in-house and by our customers.
I’ve included a graph that corresponds to Fab-ZAP human (IT-51). Here is a cytotox which tests Fab-ZAP human with Trastuzumab on SK-BR-3 cells (breast cancer cell line).
SK-BR-3 cells were plated at 1000 cells/90 μl/well and incubated overnight. Trastuzumab and Saporin dilutions were made in cell media and 10 μl was added to each well. The Trastuzumab was also diluted in cell media containing, at a final concentration, 4.5 nM/10 μl Fab-ZAP, and 10 μl was added to each well. The plates were incubated for 72 hours. The plates were developed using a solution of XTT/PMS and read at 450 nm. Cytotoxicity was analyzed by comparing well readings of the treated wells to those of the control wells, expressed as a percentage. The number of viable cells remaining on the day of development is measured via cell metabolism of a colorimetric molecule within the developing reagents. Analysis was performed using Prism software (GraphPad, San Diego).
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ZAP conjugates – These are non-targeted saporin conjugates that “piggyback” on to your primary targeting agent (biotinylated material or antibody) to eliminate specific cells and reveal cell function.
