Technical Frequently Asked Questions (FAQs)

Get your common technical support frequently asked questions (FAQs) answered by our team of scientists.

General

Yes, the online datasheets are updated as soon as we obtain additional data, so they will always have the most up-to-date information about a product. If you find any discrepancies, or require any clarification, please contact us.

 

We update the product datasheets with new publications that used a product as soon as they are available. Product citations can be found at the bottom of the each product datasheet.

 

All of our products are shipped with a datasheet that specifies how the product should be stored. Please follow the storage directions for each particular product, as we cannot guarantee the proper performance of the product if it was not stored as recommended.

Antibodies

The concentration of our purified monoclonal and polyclonal antibodies is listed on the product datasheet sent with the product and online. The concentration of unpurified polyclonal and ascites monoclonal antibodies cannot be determined as the antiserum contains proteins other the antibody.

We highly recommend that every user determine the optimal concentration of antibody needed for their particular application and tissue type. The recommended starting dilution for our antibodies is listed on the product datasheet.

If we have not yet listed a recommended starting dilution for your particular application, here are some general guidelines to get you started:

Purified Antibody Antiserum Ascites Fluid
WB 1:1000 (1 µg/ml) 1:1000 1:1000
IHC/ICC 1:500 (2 µg/ml) 1:100 1:100
ELISA 1:10000 (0.1 µg/ml) 1:500 1:10000
FACS/FCS 1:1000 (1 µg/ml) 1:500 1:1000
IP 1:1000 (1 µg/ml) 1:100 1:100

We highly recommend that you aliquot your antibody into one-time use vials in order to avoid freeze-thaw cycles. Therefore determine how much antibody you will need for one experiment, and then aliquot accordingly. Keep in mind that aliquots with less than 5-10 µl of volume may suffer more from evaporation, resulting in a large change in the stock concentration.

The concentration of PBS in our buffered antibodies is 0.01M (10mM).

The epitope (region within the immunogen that the antibody binds) is rarely mapped for antibodies, therefore unless it is listed on the datasheet under the “Immunogen” field, it is unavailable.

Whenever possible we will provide the immunogen sequence used to generate an antibody on the product datasheet. Often times the sequence is proprietary, therefore we are unable to disclose the sequence.

If you are unable to find an antibody that has been tested for use in your species, then you will need to identify an antibody that is most likely to be reactive in your species.

To do this you will need to perform an amino acid sequence alignment using the immunogen sequence used to generate the antibody, and the sequence of your target protein.

We recommend using Uniprot (http://www.uniprot.org/) to find and align your sequences. This will output the percentage of alignment. Over 80% alignment provides a good indication that the antibody may cross-react with your species.

StressMarq does not guarantee that an antibody will work in an untested species. If you would like to test a sample size of an antibody on an untested species, please checkout our FREE SAMPLES.

 

A clone number is assigned to a monoclonal antibody that is produced by a single line of hybridoma cells. It is a unique identifier that is assigned by the manufacturer of the hybridoma clone. It is separate from a lot/batch number in that one clone can have many lots/batches.

The certificate of analysis is a quality control document that validates that a product has met the standards set out by the product datasheet. It often includes data proving that a product has been tested to work under specific conditions.

First you need to choose a secondary antibody that is against the host of your primary antibody. So if your primary antibody is a Mouse Anti-HSP70 antibody, then you need to choose a secondary that is made against mouse (ie. Goat Anti-Mouse). Then from there you can choose which tag you would like to use to visualize your antibody.

Conjugates

When choosing the right fluorescent conjugate, you need to ask yourself a few questions:

  1. What filters/lasers are on your microscope?

First you need to know what filters and/or lasers you have on your microscope. They will determine what fluorescent proteins you will be able to excite and visualize.

  1. How many fluorophores do you plan to use on the one sample?

If you are using more than one fluorescent label in your sample, then you will need to carefully choose your other fluorescent dye(s) so as to minimize spectral overlap.

You can do this by looking at the emission and excitation spectra for your fluorescent dyes, and choosing pairings that reduce how much overlap the emission and excitation spectra have with others dyes.

If you are using a laser to excite your fluorophore, you only need to make sure that your laser (ie. 488 nm) is not exciting more than 2 dyes at the same time. If the laser is hitting the leading edge of your second fluorescent dye excitation spectra, then you may end up with a slight excitation of that dye.

However if the filter that you are using to capture the emission spectra of that dye does not pick up any of the emission spectra of the second dye, then you will not see any spectral overlap while capturing your images.

The conjugate is bound to the antibody via a covalent bond, which is highly stable.

Conjugated antibodies can be labelled with anywhere from 1-10 tags depending on the antibody being conjugated and the tag being applied.

The conjugate tags bind to the antibody via free amine groups. It is most likely that the tag will bind in the Fc region of the antibody, as there are more free amine groups present in that area.

However, it is possible that the tag may bind in the paratope of the antibody, thereby limiting binding of the antibody to the antigen. Although this is unlikely, it could affect the ability of the antibody to bind to the antigen in all applications.

As we cannot control the binding of the conjugate to the antibody, there is no way to confirm or guarantee the location of the antibody tag.

All conjugated primary antibodies with a buffer containing 50% glycerol can be stored at -20°C. Conjugated antibodies without 50% glycerol should be stored at 4°C.

Conjugated primary antibodies are stable for up to 18 months when stored at 4°C, and up to 2 years when stored at -20°C. Please see “How should a conjugated primary antibody be stored?” for further storage information.

In rare cases, the conjugate tag may bind in the paratope of the antibody, thereby limiting binding of the antibody to the antigen. Although this is unlikely, it could affect the ability of the antibody to bind to the antigen in all applications.

As we cannot control the binding of the conjugate to the antibody, there is no way to confirm or guarantee the location of the antibody tag.

Proteins

The activity of all of our proteins is listed on the specific product datasheet.

Endotoxin-free proteins are labeled on the specific product datasheet.

Small Molecules

All of our small molecules have solubility information listed on their datasheet. Please follow the specific instructions for selecting a solvent.

No, none of our small molecules are sterile. If you are reconstituting it in water or PBS, then you would need to perform sterile filtration. However if you are reconstituting it in an organic solvent (DMSO or ethanol), then sterile filtration is not needed as the solvent would kill any microorganisms.

 

Kits

It takes a great deal of time and effort to correctly match pairs of antibodies for any immunoassay, therefore we do not provide details as to which antibodies were used to make our kits.

The pre-treatment buffer included in some of our ELISA kits is used to dissociate the target protein from other proteins it could be associated with.

 

The sensitivity of an ELISA kit is calculated as follows:

{2(StDev of blank) (Ave concentration of lowest standard above blank)} / (Aver. OD of lowest standard above blank- Aver. OD of blank).  Will need 10-20 samples for this calculation.

The limit of quantification is equal to the lowest concentration of protein used in the standard curve.