Frequently Asked Questions

FlexAble Antibody Labeling Kit

FlexAble

 

Q: What are the FlexLinker, FlexQuencher and FlexBuffer?
A: The FlexLinker is a small polypeptide to which dyes are covalently conjugated that can label unconjugated primary antibodies. 
The FlexQuencher is an Fc-containing fragment that neutralizes the excess FlexLinker.
The FlexBuffer is a PBS-based buffer.

 

Q: Do I need to aliquot the FlexLinker, FlexQuencher and FlexBuffer upon receipt?
A: Aliquoting is optional. FlexAble kits remain active through 50 freeze-thaw cycles.

 

Q: Is it fine to freeze-thaw the reagents 50 times?
A: FlexAble kits remain functional and the signal remains robust through freeze-thaw cycles. Although aliquoting may increase shelf life.

 

Q: What is the recommended storage of the kit?
A: We recommend to store at -20°C for up to 1 year after receipt. Storage at +4°C is possible for 6 months.

 

Q: What is the minimal quantity I can label?
A: You can label as little as 0.5 µg primary antibody with one labeling reaction.

 

Q: What is the largest quantity I can label?
A: With a standard kit size (50 reactions), you can label 25 µg of one antibody or up to 50 different antibodies. You can easily scale up the antibody amount per labeling approach.

 

Q: How do I scale up the FlexLinker, FlexBuffer, and FlexQuencher if I want to label larger quantities of an antibody?
A: You can scale up the required volumes proportionally according to the amount of primary antibody required. See the table below for example calculations:
Antibody quantitiy
0.5 µg
1 µg
2.5 µg
5 µg
10 µg
25 µg
FlexLinker Volume
1 µL
2 µL
5 µL
10 µL
20 µL
50 µL
FlexBuffer Volume
≤ 8 µL
≤ 16 µL
≤ 40 µL
≤ 80 µL
≤ 160 µL
≤ 400 µL
FlexQuencher Volume
2 µL
4 µL
10 µL
20 µL
40 µL
100 µL

 

Q: What is the lowest concentration of my primary antibody that I can use?
A: Our protocol uses 0.5 µg of primary antibody in 7 µL, which ends up at 0.07 mg/mL. If the concentration of your antibody is lower, you can also use a larger volume than 7 µL.

 

Q: What is the highest concentration of my primary antibody that I can use?
A: There is no limitation for high concentrations of primary antibodies. If the concentration is very high and the volume to be pipetted is too small, you can dilute the antibody in PBS buffer to 0.5-1 mg/mL.

 

Q: What should I do when I don't know the concentration of my primary antibody?
A: Below is a list of common antibody sources and concentrations you can use to estimate the concentration of your primary antibody:
  • Polyclonal antibody from serum: approx. 1 mg/mL specific antibody, approx. 10 mg/mL total antibody
  • Monoclonal antibody from tissue culture supernatant (with 10% FBS): approx. 0.05 mg/mL specific antibody, approx. 1 mg/mL total antibody
  • Monoclonal antibody from tissue culture supernatant (serum-free media): approx. 0.05 mg/mL specific antibody, approx. 0.05 mg/mL total antibody
  • Monoclonal antibody from ascites: 0.9-9 mg/mL specific antibody, 1-10 mg/mL total antibody
(From "Antibodies: A Laboratory Manual", E. Harlow & D. Lane,  CSHL Press 1988)

 

Q: Can I label primary antibodies stored in BSA, glycerol, Tris buffer and/or preservatives?
A: Yes, FlexAble Antibody Labeling Kits have been validated with carriers and amine buffers. Neither BSA nor amine buffers, in any chosen concentration, interfere with the labeling. 50% glycerol as well as preservatives like sodium azide are also compatible with the kit.

 

Q: How much antibody do I need to label for an experiment?
A: Depending on the experiment and strength of your primary antibody, we suggest the following:
  • Immunofluorescence: 0.05 to 0.5 µg per experiment (~5 samples from one labeling reaction)
  • Flow Cytometry: 0.2 to 2.0 µg per experiment (~2 samples from one labeling reaction)
  • Western Blot: 0.5 to 5.0 µg per experiment (max. 1 blot per one labeling reaction)

 

Q: How many different primary antibodies can I label with one kit?
A: You can label up to 50 different antibodies with our FlexAble 50 rxn Kit, and up to 10 antibodies with our FlexAble 10 rxn Kit.

 

Q: What is the degree of labeling (DOL) achieved by FlexAble Kits?
A: FlexAble labels each primary antibody with 2 dyes.

 

Q: Will I observe cross-reactivity/leaking when I use two FlexAble-labeled antibodies from the same species during multiplexing?
A: FlexAble labels primary antibodies with a high affinity FlexLinker. Dissociation of FlexLinker from one antibody and association to another antibody is rare. If you observe leaking, we recommend adding more FlexQuencher to remove unbound FlexLinker, or you can try sequential staining of the labeled antibodies.

 

Q: What can I do to decrease background?
A: You can reduce the FlexQuencher amount or increase your washes after staining.

 

Q: Do I need to add FlexQuencher to remove dyes?
A: When you only stain samples with one primary antibody or primary antibodies from different species or isotypes, FlexQuencher is not necessary.

 

Q: Do I need to remove unbound FlexLinker?
A: Unbound FlexLinker is neutralized by adding FlexQuencher. No additional removal step (e.g., by ultrafiltration) is required.

 

Q: Does FlexAble Antibody Labeling Kit for Mouse IgG1 work with other mouse primary antibodies?
A: No, FlexAble Antibody Labeling Kit for Mouse IgG1 can only label mouse IgG1 primary antibodies since our Kit is isotype specific.

 

Q: Is staining with FlexAble as sensitive as staining with unlabeled primary and fluorescent secondary antibodies?
A: Direct immunofluorescence detection with FlexAble is typically more specific with less background but may yield less total signal than indirect detection.