General Frequently Asked Questions
- Antibody Product Line
- eFluor® Nanocrystals Tools for Discovery Questions
- eFluor® Nanocrystal Conjugation Kits
- Organically-Soluble Nanocrystals
Antibody Product Line
Q. What type of antibodies do you offer?
eBioscience offers a wide range of monoclonal and polyclonal antibodies for detecting murine, human, non-human primate and other species of antigens. Antibodies can be used for the detection of cell surface markers, such as CD molecules, cytoskeletal and cytoplasmic proteins, chemokine receptors, as well as intracellular cytokines/chemokines and intra-nuclear proteins including transcription factors.
Q. What is the shelf life of your antibodies?
The majority of eBioscience antibodies have a guaranteed shelf life of one year from date of receipt unless indicated on our Technical Data Sheets. This guarantee is provided if the products are kept under optimal storage conditions as stated on the Technical Data Sheet. If you are not completely satisfied with the performance of your product, please contact technical support at tech@ebioscience.com for assistance.
Q. What information do you have regarding the cross-reactivity of your antibodies?
Information about known cross-reactivity of an antibody is given on the Technical Data Sheets. We have done some in-house testing to analyze cross-reactivity of anti-human antibodies amongst non-human primates. The results of these tests are summarized in our Cross-Reactivity Charts.
Q. What formats do you offer?
For convenience, eBioscience offers antibodies in a variety of formats, Best Protocols® Fluorescent Dyes Chart. We carry purified and biotin conjugates with and without sodium azide, and a large number of direct fluorochrome conjugates. Our newest additions are eFluor® Nanocrystals (NC) and eFluor® Organic Dyes, for more information please click here. These include eFluor 605NC, 625NC, 650NC (all nanocrystal reagents) as well as eFluor 450 (Pacific Blue® replacement), APC-eFluor 780 (APC-Alexa® Fluor 750 replacement), eFluor 660 (Alexa Fluor 647 replacement) and PerCP-eFluor 710 .
Q. What is the difference between your Affinity Purified and Functional Grade Purified formats?
The Affinity Purified format of our monoclonal antibodies contains no additional protein carriers such as BSA or gelatin but does contain sodium azide as a preservative. These products can be used for flow cytometry, WB, IP, and IHC applications. The Functional Grade purified formats contain no sodium azide and are tested for endotoxin. These products meet or exceed the industry standard of 0.001 ng endotoxin/ug antibody and in most instances have endotoxin levels significantly lower than these standards (unless otherwise stated on the product technical datasheet). Please contact tech@ebioscience.com if you would like lot specific information for Functional Grade products. The Functional Grade formats are generally used for bioassays and in vivo studies.
Q. What are the concentrations of your antibodies?
eBioscience offers both test and microgram to milligram sizes, depending on the product. The concentration of the test sizes is indicated on the Technical Data Sheets under Applications Tested. The concentrations of the microgram sizes vary depending on the format. Please refer to the product vial for lot specific information.
Q. What applications can I use eBioscience antibodies for?
eBioscience antibodies can be used for a variety of applications, such as:
- Flow cytometric analysis- surface antigen staining (FC)
- Flow cytometric analysis- intracellular staining (IC Flow)
- Immunohistochemistry (IHC)
- Immunohistochemistry- frozen sections (IH/F)
- Immunohistochemistry- paraffin sections (IHC-Paraffin)
- Immunoblotting (WB)
- Immunoprecipitation (IP)
- ELISA
- ELISPOT
- Bioassays (FA)
- Neutralization/Blocking studies (NU)
- In vitro Cytokine Capture Assay (IVCCA)
- In vivo studies in mice
- Chromatin immunoprecipitation (ChIP)
eFluor® Nanocrystals Tools for Discovery Questions
Q. Which biomolecules can be conjugated to eFluor Nanocrystals?
eFluor Nanocrystals have been successfully conjugated to peptides, proteins and oligonucleotides.
Q. Which protocol do I use for conjugation of a biomolecule to eFluor Nanocrystals?
Protocols for conjugation of either amine or carboxyl eFluor Nanocrystals are provided in our Best Protocols®. These protocols will also be provided with your order of Reactive eFluor Nanocrystals.
Q. Can I conjugate eFluor Nanocrystals to an antibody myself?
Reactive eFluor Nanocrystals, in a variety of emission spectra, with both carboxyl and amine surface chemistries are available for conjugation. Conjugation protocols are available in our Best Protocols and are provided when you order Reactive eFluor Nanocrystals. For more details, please see our Tools for Discovery product listing.
eFluor® Nanocrystal Conjugation Kits
Q. Which kit should I choose: ‘Amine-Reactive’ or ‘Sulfhydryl-Reactive’?
The most important factor in choosing the kit is knowing which functional group is available on your biomolecule. If your molecule has a primary amine, then you should choose the ‘Amine-Reactive’ Kit. If your molecule has a sulfhydryl group (thiol, disulfide, or cysteine residue), then you would choose the ‘Sulfhydryl-Reactive’ Kit. Antibodies, for example, have both functional groups available, so you can choose either kit.
Q. Do both Sulfhydryl-Reactive and Amine-Reactive kits perform equally well in all applications?
We have not tested all applications. Both kits have been extensively tested in flow cytometry, immunocytochemistry (ICC) and immunohistochemistry (IHC). Although both kits give good results in these applications, we have found that the Sulfhydryl-Reactive kit may demonstrate a slight advantage for flow cytometry applications, and the Amine-Reactive kit may demonstrate a slight advantage for ICC/IHC applications but this will vary depending on the particular protein. It is critical for optimal performance to use the recommended protocols and buffers as stated in our Best Protocols.
Q. How many functional groups are there on each eFluor Nanocrystal?
There are an estimated 30–60 functional reactive groups on each eFluor Nanocrystal.
Q. How much eFluor Nanocrystal is included in each kit?
The kits are optimized for the conjugation of 200 ug of antibody per reaction at a ratio of 2:1 (for the eFluor 605NC) and 4:1 (for the eFluor 650NC) antibody to eFluor Nanocrystal. Therefore, each reaction in the kit contains 0.67 nmol of reactive eFluor Nanocrystal for the eFluor 605NC and 0.33 nmol for the eFluor 650NC.
Q. What types of proteins can I conjugate?
Please refer to Other Proteins section.
Q. I have finished conjugating my biomolecule to the eFluor Nanocrystal, can I freeze the conjugate for long-term storage?
No, freezing eFluor Nanocrystal conjugates is detrimental to their performance. eFluor Nanocrystal conjugates should be stored at 2-8°C in the dark. We recommend storing your vials away from the back of the refrigerator where samples may come close to freezing, which will affect performance.
Q. I followed your protocol exactly to conjugate my antibody to the eFluor Nanocrystals, but I see no signal in my application. What should I do?
Certain antibodies may perform better using one chemistry over the other. For example, if an amine group is present in the antigen binding site, conjugation with the Amine-Reactive conjugation kit to this amine may block binding of the antibody to its epitope. Alternatively, if the disulfide linkages in your antibody are blocked or protected, the Sulfhydryl-Reactive kit will not work because conjugation to the eFluor Nanocrystals will not occur. In either case, the best solution is to try the alternative kit chemistry. Please see the section(s) below concerning the application you are performing for additional troubleshooting assistance.
Q. Does it matter what my final conjugation volume and antibody concentration is once I add the antibody to the eFluor Nanocrystal solution?
The eFluor Nanocrystal Conjugation Kits are optimized for a total conjugation volume of 300 uL. However, you may achieve slightly better results if the conjugation volume is kept at approximately 100 uL total conjugation volume. Conjugation volumes as high as 550 uL have been tested, showing that the larger conjugation volume may result in a slight reduction in performance of the final conjugate.
Q. Do I need to perform the conjugation reaction in the dark?
No, there is no significant difference between eFluor Nanocrystal conjugations done in ambient light as compared to in the dark.
Q. Do I have to conjugate my molecule for the full 2 hours?
We have tested antibody conjugation times as short as 30 minutes without any significant reduction in performance in either the Sulfhydryl-Reactive or Amine-Reactive Kits. However, we recommend a 2 hour conjugation time as stated in the protocol to accommodate biomolecules that may have kinetically-slower reaction rates (for example, if the sulfhydryl or amine groups are sterically-hindered). Conjugation times longer than 2 hours do not increase the conjugation efficiency.
Q. Does it matter if I allow the quenching reaction to go longer/shorter than the time listed in the protocol?
Yes, the quenching step is one of the most time-sensitive steps in the eFluor Nanocrystal Conjugation Kit procedure. We highly recommend keeping the quenching reaction to 10 minutes as stated in the protocol. Shorter quenching times may increase background issues associated with non-specific binding, and longer quenching times may decrease the fluorescence intensity of the eFluor Nanocrystal Conjugated protein.
Q. What is the shelf-life of my eFluor Nanocrystal conjugate?
This may vary depending on the type of biomolecule attached, but we always recommend using the conjugate immediately for best results. However, conjugates are stable for 4-6 months after conjugation when stored properly (Stored at 2-8°C in the dark).
Antibody
Q. My biomolecule is in a buffer containing sodium azide. Will this interfere with the conjugation?
We have verified that sodium azide concentrations up to 0.09% (w/v) do not interfere with either the Sulfhydryl-Reactive or the Amine-Reactive eFluor Nanocrystal Conjugation Kits.
Q. My biomolecule is in a high/low pH buffer, will this affect the conjugation reaction?
Yes, if the biomolecule is present in a buffer outside the pH range (7-9 for the Amine-Reactive kit and 6.5-7.5 for the Sulfhydryl-Reactive kit), you should buffer exchange it into a buffer such as PBS or Borate in the pH range from 6-8, and concentrate it into 100 – 300 uL total volume prior to use.
Q. My antibody is in a buffer containing carrier proteins such as serum or BSA. Will this interfere with the conjugation?
Yes, carrier proteins will interfere with both the Amine-Reactive and Sulfhydryl-Reactive eFluor Nanocrystal Conjugation Kits. We suggest that you purify your antibody on a Protein G or Protein A resin and resuspend in PBS or TBS buffer.
Other Proteins
Q. What if I want to conjugate a molecule that is not an antibody?
When conjugating new biomolecules to eFluor Nanocrystals, it is recommended to perform a titration to verify and optimize the performance of the conjugate. Other biomolecules should conjugate to the eFluor Nanocrystal provided that the biomolecule contains the appropriate functional group for conjugation, (free sulfhydryl or disulfide bridge for the Sulfhydryl-Reactive kit, or a primary amine for the Amine-Reactive kit) and the molecular weight is between 30-150 kDa (Amine-Reactive kit) or less than 150 kDa (Sulfhydryl-Reactive kit).
Q. My biomolecule has a molecular weight greater than 150 kDa. Will the conjugation kit work for me?
Although the conjugation will still likely occur, the purification of molecular weights greater than 150 kDa using our eFluor Nanocrystal Kits is unlikely. You can still use the kit(s), but you will have to purify the conjugate from un-reacted biomolecules using a technique capable of resolving the two molecular weights. Effective techniques for separation include size-exclusion chromatography, ion-exchange chromatography, or hydrophobic interaction chromatography.
Q. My biomolecule has a molecular weight lower than 30 kDa. Will the conjugation kit work for me?
For the eFluor Nanocrystal Sulfhydryl-Reactive kit, this will not be a problem. During the protocol for the Amine-Reactive kit, however, your modified biomolecule will not elute from the spin column, therefore molecules with low molecular weights cannot be used with the Amine-Reactive kit at this time.
Q. My biomolecule is in a buffer containing sodium azide. Will this interfere with the conjugation?
We have verified that sodium azide concentrations up to 0.09% (w/v) do not interfere with either the eFluor Nanocrystal Sulfhydryl-Reactive or Amine-Reactive eFluor Nanocrystal Conjugation Kits.
Q. My biomolecule is in a high/low pH buffer, will this affect the conjugation reaction?
Yes, if the biomolecule is present in a buffer outside the pH range of 6-8, you should buffer exchange it into a buffer such as PBS or Borate in the pH range (7-9 for the Amine-Reactive kit and 6.5-7.5 for the Sulfhydryl-Reactive kit), and concentrate it into 100 – 300 uL total volume prior to use.
Sulfhydryl-Reactive Kit Protocol Questions
Q. What if I don’t have a 60°C water bath?
Take a 500 mL glass beaker and fill it with 250 mL H2O. Either heat the beaker in a microwave, or place it onto a hot plate until the temperature reaches ~60°C. If the temperature gets too hot, add cool tap water to adjust.
Q. My molecule has a disulfide, not a sulfhydryl functional group. Will that still conjugate using the eFluor Nanocrystal Sulfhydryl-Reactive kit?
Yes, free sulfhydryl groups as well as disulfide bonds and cysteine groups may be used to conjugate to the Sulfhydryl-Reactive eFluor Nanocrystal Conjugation Kit.
Q. Do I need to reduce my molecule using B-ME or DTT prior to use?
No, you do not have to reduce your molecule. You can simply add the molecule to the reconstituted eFluor Nanocrystals and proceed with the conjugation.
Q. How will the Sulfhydryl-Reactive kit affect the function of my protein?
The eFluor Nanocrystal Sulfhydryl-Reactive kit will reduce any disulfides to free sulfhydryl functional groups and then form a covalent bond to the eFluor Nanocrystal outer coating. If your protein has structural components or activity that is dependent upon the disulfide bond, then you may achieve better results with the eFluor Nanocrystal Amine-Reactive kit.
Q. How does the Sulfhydryl-Reactive kit work?
The Sulfhydryl-Reactive kit contains an eFluor Nanocrystal formulation containing a unique reducing agent and maleimide-functionalized nanocrystals. The reduced sulfhydryl group from the biomolecule conjugates in-situ to the eFluor Nanocrystal on the outer coating.
Q. Do I have to conjugate my molecule for the full 2 hours?
We have tested antibody conjugation times as short as 30 minutes without any significant reduction in performance in either the Sulfhydryl-Reactive or Amine-Reactive Kits. However, we recommend a 2 hour conjugation time as stated in the protocol to accommodate biomolecules that may have kinetically-slower reaction rates (for example, if the sulfhydryl or amine groups are sterically-hindered). Conjugation times longer than 2 hours do not increase the conjugation efficiency.
Q. Does it matter if I allow the quenching reaction to go longer/shorter than the time listed in the protocol?
Yes, the quenching step is one of the most time-sensitive steps in the eFluor Nanocrystal Conjugation Kit procedure. We highly recommend keeping the quenching reaction to 10 minutes as stated in the protocol. Shorter quenching times may increase background issues associated with non-specific binding, and longer quenching times may decrease the fluorescence intensity of the eFluor Nanocrystal Conjugated protein.
Amine-Reactive Kit Protocol Questions
Q. If I use a lower antibody concentration (0.5 mg/mL in 400 uL) for the Amine-Reactive kit, will additional steps/supplies be required?
Yes, because each spin column provided in the kit can only de-salt a volume of 100 uL, you will either need 4 spin columns (2 are provided) to de-salt the activated antibody solution (400 uL), or you will need to concentrate the antibody solution down to 100 uL and use a single spin column, as detailed in the protocol.
Q. How will the Amine-Reactive kit affect the function of my protein?
The Amine-Reactive kit will modify available primary amine groups on your protein and then conjugate to the eFluor Nanocrystal surface through those modifications. If the active portion of your biomolecule is known to contain primary amine functionalities, conjugation of the eFluor Nanocrystals to these amines could block activity of your biomolecule. In this instance, you may achieve better results with the Sulfhydryl-Reactive eFluor Nanocrystal kit.
Q. How does the Amine Reactive kit work?
The Amine-Reactive kit will first activate available primary amine functional groups on your biomolecule with a hydrazine moiety. Hydrazine is specific for a coupling reaction with our formylbenzamide-modified eFluor Nanocrystals.
Q. Do I have to conjugate my molecule for the full 2 hours?
We have tested antibody conjugation times as short as 30 minutes without any significant reduction in performance. However, we do recommend a 2 hour conjugation time as stated in the protocol to accommodate biomolecules that may have kinetically-slower reaction rates.
Q. Does it matter if I allow the quenching reaction to go longer/shorter than the time listed in the protocol?
Yes, the quenching step is one of the most time-sensitive steps in the eFluor Nanocrystal Conjugation Kit procedure. We highly recommend keeping the quenching reaction to 10 minutes as stated in the protocol. Shorter quenching times may increase background issues associated with non-specific binding, and longer quenching times may decrease the fluorescence intensity of the eFluor Nanocrystal Conjugated protein.
Questions about Applications for eFluor® NC conjugated molecules
Q. Do both Sulfhydryl-Reactive and Amine-Reactive kits perform equally well in all applications?
We have not tested all applications. Both kits have been optimized for flow cytometry as well as immunocytochemistry (ICC) and immunohistochemistry (IHC). Although both kits generate good results in these applications, we have found that the Sulfhydryl-Reactive kit may demonstrate a slight advantage for flow cytometry applications, and the Amine-Reactive kit may demonstrate a slight advantage for ICC/IHC applications but this will vary depending on the particular protein. It is critical for optimal performance, to use the recommended protocols and buffers as stated in our Best Protocols.
Also refer to eFluor® Nanocrystal FAQs
Questions about Organically-Soluble Nanocrystals
Q. What are “organically-soluble” nanocrystals? How are these nanocrystals different from the other nanocrystals sold by eBioscience (amine, carboxylate, etc.)?
“Organically-soluble” nanocrystals are nanocrystals that have their native, out-of-reaction surface ligands still attached to their surface. They are the precursor form of nanocrystal to our water-stable nanocrystal offerings (i.e. the organically-soluble 650NC is used to make the amine-functionalized, carboxylate-functionalized, and non-functionalized 650NC nanocrystals).
The difference between an organically-soluble nanocrystal and our other nanocrystal offerings has to do with the outer layer of molecules surrounding the nanocrystal; the underlying nanocrystal itself is exactly the same. The organically-soluble nanocrystals have a coordinating layer of trioctylphosphine oxide (TOPO) ligands passivating the outer shell of the nanocrystal; while the amine, carboxylate, or non-functional nanocrystals have an additional lipid micelle layer containing polyethylene glycol (PEG) as the outermost molecular layer. Because TOPO ligands are hydrophobic, the organically-soluble TOPO-capped nanocrystal will only disperse into organic solvents, whereas the PEG-capped nanocrystals are hydrophilic and will disperse into aqueous solvents.
Q. What solvents are organically-soluble nanocrystals soluble in?
The organically-soluble nanocrystals that we sell are dispersed in toluene. Other common solvents for these nanocrystals include chloroform, hexanes, and pyridine (as well as others.) They are not soluble in water, methanol, ethanol, DMSO, etc.
Q. What can I do with organically-soluble nanocrystals?
Good question! We realize that the lipid micelle coating on our amine, carboxylate, or non-functionalized nanocrystals may be limiting to different research applications in the life sciences. In order to provide some flexibility, we offer the organically-soluble nanocrystals (which are the precursor form of nanocrystal to the water-stabilized version) as a means to enable the use of custom water-stabilizing coatings. These organically-soluble nanocrystals are helpful, for example, if our lipid micelle-coated nanocrystals are too large (~20 – 25 nm hydrodynamic diameter), do not offer the desired functional group (amine, or carboxyl), or may not have the desired charge (cationic or anionic) for particular applications. By offering the organically-soluble nanocrystals, any of these potential limitations may be overcome by the researcher. Below are just a few references detailing a few of the myriad of custom methods for modifying organically-soluble nanocrystals for aqueous stability:
- Dubertret B. Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchaber A. In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science. 2002; 298(5599): 1759-1762. [Phospholipid Micelles, (PubMed)]
- Anderson RE, Chan WC; Systematic Investigation of Preparing Biocompatible, Single, and Small ZnS-Capped CdSe Quantum Dots with Amphiphilic Polymers. ACS Nano. 2008; 2(7): 1341. [Amphiphilic Polymers, (PubMed)]
- Smith A.M. Minimizing the Hydrodynamic Size of Quantum Dots with Multifunctional Multidentate Polymer Ligands. JACS 2008; 130(34): 11278-11279. [Multidentate Polymers, (PubMed)]
- Uyeda HT, Medintz IL, Jaiswal JK, Simon SM, Mattoussi H.; Synthesis of Compact Multidentate Ligands to Prepare Stable Hydrophilic Quantum Dot Fluorophores. JACS 2005; 127(11): 3870-3878. [Multidentate Ligands, (PubMed)]
- Stewart MH, Susumu K, Mei BC, Medintz IL, Delehanty JB, Blanco-Canosa JB, Dawson PE, Mattoussi H.; Multidentate Poly(ethylene glycol) Ligands Provide Colloidal Stabiilty to Semiconductor and Metallic Nanocrystals in Extreme Conditions. JACS 2010; 132(28): 9804-9813. [Multidentate PEG, (PubMed)]
Q. How long are these organically-soluble nanocrystals stable?
The as-sold vials of organically-soluble nanocrystals arrive in anhydrous toluene and are stable for up to 6 months if left unopened and stored at room temperature. Water condensation is likely the biggest contributor to nanocrystal degradation, therefore storing the vial at 4°C and/or opening the vial numerous times will decrease the product shelf life.
Q. Can these organically-soluble nanocrystals be applied in the same way as other nanocrystalsi.e. can they be used for flow/imaging/microscopy?
Once modified for aqueous stability and attached to a biological molecule, these organically-soluble nanocrystals may be used in similar assays as our other nanocrystal product offerings.
Q. Is toluene harmful to cell preparations?
Toluene may be harmful to cell preparations if not removed completely. We recommend good purification techniques such as size-exclusion chromatography or ultracentrifugation following surface modification of the nanocrystal to remove not only excess coating molecules, but residual organic solvent as well.
Q. Are these organically-soluble nanocrystals soluble in water?
No, these organically-soluble nanocrystals are completely insoluble in water.
Q. How can I modify these organically-soluble nanocrystals to disperse in water?
Organically-soluble nanocrystals may be modified for water compatibility by either replacing the native TOPO ligands with a hydrophilic molecule (such as mercaptoacetic acid), or using an amphiphilic molecule. Numerous modification methods have been reported in the literature. Please see the references listed above.
Q. Can I conjugate organically-soluble nanocrystals to my antibody/biomolecule?
After surface coating of the organically-soluble nanocrystal for aqueous stability and the introduction of a functional group on the surface of the coating layer (such as an amine, carboxylate, maleimide, etc.), the nanocrystals may be conjugated to biomolecules.
Q. I modified my organically-soluble nanocrystal with a water-stable coating, and the fluorescence emission changed. What’s happening?
Depending on the route of surface modification, the nanocrystal’s emission properties may be expected to change in a variety of ways. Coatings that form covalent attachment to the physical surface of the nanocrystal (such as mercapto-alkyl acid molecules) will likely have the largest effect on the emission properties by possibly red-shifting the photoluminescence and/or decreasing the quantum efficiency (brightness) of the nanocrystal. Other coating methods using amphiphilic molecules that do not displace the native TOPO ligands on the nanocrystal surface may effect the emission properties much less because they do not directly interact with the nanocrystal. The end results of the nanocrystal’s emission properties will be highly dependent upon many different properties of the water-stabilizing coating.