Primary Antibodies Frequently Asked Questions topic (I)

2022-08-05 Hits(568)

Q1: Is the use of a Golgi blocker recommended prior to intracellular staining for secreted cytokines?

 

Answer:

Generally 3 uM Monesin is used in combination with cell stimulation to prevent signal loss.

 

Q2:Are KMD Systemrecombinant proteins and antibodies sterile?

 

Answer:

Although the vials are bottled using aseptic techniques, heat-treated vials, and sterile stock solutions, they are not considered or guaranteed to be sterile. If sterile material is needed for an experiment, the material can be filtered through a 0.2 micron filter designed for use with biological fluids.

 

Q3:Can I use your antibodies for a matched pair ELISA with a standard from another company?

 

Answer:

Yes, but there are potential issues to consider. There can be differences in immunological recognition or the binding between antibody and recombinant protein. This can happen if there is a difference in the folding or sequence of the recombinant protein used as the standard versus the recombinant protein used as the immunogen for the antibody. Protein folding or sequence differences in the antibody-binding region can lead to poor (or no) recognition of the standard.

 

Q4:Does trehalose interfere with metal conjugation to antibodies, for CyTOF?

 

Answer:

Trehalose is not reported to interfere with metal conjugation of antibodies, for CyTOF.

 

Q5:For Antigen-Affinity Purified antibodies, does the antibody eluted from an affinity purification column still have any of the affinity ligand associated with it and is there any procedure to separate that ligand from the antibody? 

 

Answer:

There has been no evidence of ligands leaching off the affinity columns used.  The ligand is covalently bound to the resin and remains on the resin when the antibody is eluted.

 

Q6:What is a direct ELISA?

 

Answer:

In a direct ELISA, a plate is coated with the analyte of interest and a labeled detection antibody is used to verify the presence of the analyte. The direct ELISA may use a colorimetric, chemiluminescent, or fluorescent reporter.

 

Q7:What epitope does this polyclonal antibody recognize?

 

Answer:

Polyclonal antibodies generally recognize multiple epitopes because they are generated using the entire immunogen stated on the product datasheet.

 

Q8:What is trehalose and why is it in the formulation?

 

Answer:

Trehalose is a non-reducing sugar and does not react with amino acids or proteins as part of the Maillard reaction. It is found in nature in many plants and animals. Trehalose is an effective sugar for stabilizing proteins against damage caused by freezing. It can also make the protein more resistant to moisture when lyophilized, resulting in a product that is less likely to precipitate when reconstituted.

 

Q9:Will trehalose affect my conjugation reaction?

 

Answer:

It is possible that the presence of trehalose will interfere in the successful conjugation of a protein. This will depend on the method used, and the customer should investigate this potential prior to purchasing the product.

 

Q10:Will trehalose affect the performance of the protein or antibody in my specific application?

 

Answer:

We have seen no adverse effect in our bioassays or other approved applications. However, customers are advised to run a control in their assay to determine if the concentration of trehalose in the protein or antibody formulation has any adverse effects.

 

Q11:Can cells be stained for flow cytometry and analyzed the following day?

 

Answer:

In most cases, stained cells can be analzyed the following day if they are fixed and stored appropriately. 

 

Q12:The fluorescent-conjugated antibody datasheet lists applications other than flow cytometry in the Specificity section. Does this antibody have further utility in those applications?

 

Answer:

The Specificity section describes the characterization of the unconjugated antibody in applications such as Western blot and Direct ELISA. Cross-reactivity with other species and related molecules is described, as tested.  The fluorescent-conjugated antibody datasheet features flow cytometry data with natural samples.  Please see the Related Products & Information tab for a convenient link to related antibodies evaluated for use in other applications. 

 

Q13:How to select isotype control?

 

Answer:

The primary purpose of the isotype control was to determine that binding of the primary antibody was specific and not nonspecific for Fc receptors or interactions with other proteins. The isotype control should be identical with the source, Ig typing and marker of the primary antibody.

 

Q14:Why are the actual detected WB bands different from those expected?

 

Answer:

WB separates proteins based on their size, and usually small molecular weight proteins migrate faster in gels. However, the mobility is also affected by other factors, which causes the difference between the actual detected band and the expected band.