We’re highlighting one of our antibodies, a rabbit polyclonal against Melanopsin. This antibody is offered as both serum (AB-N38) and affinity purified (AB-N39). Intrinsically photosensitive retinal ganglion cells (ipRGCs) are cells that express melanopsin. These ipRGCs, with their long processes, are involved in the perception of light and dark and are circadian rhythm determinants. Our Melanopsin antibody recognizes a portion of the N-terminal region of the mouse melanopsin extracellular domain and does not cross-react with melanopsins of other species, so it’s specific to mouse.
Here is how other researchers are using them in recent publications. This Melanopsin antibody may be the antibody your lab needs.
Kim et. al. used (AB-N38) at a 1:2,000 dilution to quantify ipRGCs in the retina and confirm that Per1-deficiency did not affect melanopsin-positive cell abundance.
And finally, Zhu et. al. using affinity purified (AB-N39) at a 1:2000 dilution to identify and quantify ipRGCs in retinal whole-mounts following ischemic injury.
I am using your Fab-ZAP Human (IT-51) and I want to know how many antibody molecules can a secondary antibody conjugate theoretically bind to?
Answer
A whole IgG (bivalent) secondary antibody in theory is capable of binding two antigen molecules. Since our Fab-ZAP conjugates actually use a monovalent secondary antibody, in theory they would be able to each bind one antigen molecule. Our Fab-ZAP human conjugates use polyclonal monovalent secondary antibodies raised against both heavy and light chain of IgG and can cross-react across immunoglobulin classes and subclasses of the same species since they share the same light chain. In theory it would be possible that your primary antibody could have several secondary conjugates attached.
Question
When exploring the usage concentration for Fab-ZAP Human, what is the recommended concentration range (for example, is it a few times the initial concentration of the analyte antibody / the highest test concentration)?
Answer
Our standard protocol (in vitro only) for our Fab-ZAP secondary conjugates is to maintain a constant concentration of 4.5 nM of the Fab-ZAP while titrating the primary antibody, usually in a range starting at 10 nM with 1:5 to 1:10 serial dilutions. The goal would be to have the primary antibody as a ‘variable’ and the Fab-ZAP as the ‘constant’. Also, it’s important to saturate the primary antibody with Fab-ZAP secondary conjugate, as any ‘free unreacted primary antibody’ would compete with ‘antibody+Fab-ZAP conjugate’ for binding sites on the cell. I have attached a publication that discusses this topic.
How is a targeted toxin able to inhibit a cellular process when an antagonist did not?
An antagonist is used to block a receptor on a cell to keep it from binding a target molecule and activating the cell.
For example, a substance P antagonist binds to the substance P (NK-1) receptor. The hypothesis was that if the antagonist binds to the receptor, substance P can’t bind and the cell won’t be activated.
The reality is that there are other receptors besides the substance P receptor on that cell. If any of these other receptors bind to their target molecules, then the cell will still be activated.
The Targeted Toxins with our targeting technology can use any of these cell surface receptors to target and completely eliminate the entire cell. Since there are no receptors left to bind; no cell left to be activated.
Importantly, our conjugates cleanly remove one particular cell type and don’t damage bystander cells.
Once the debris from the targeted cell is cleared away, there is nothing remaining to interfere or affect the normal action/interaction of other cells.
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
