Frequently Asked Questions
FlexAble Antibody Labeling Kit
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 a recombinant IgG or Fc fragment that neutralizes the excess FlexLinker.
The FlexBuffer is a PBS-based buffer.
Q: What is the difference between FlexAble and FlexAble 2.0 Antibody Labeling Kits?
A: FlexAble 2.0 Antibody Labeling Kits are the newest, improved version of our original FlexAble Antibody Labeling Kits. FlexAble 2.0 has 5 fluorophores conjugated to each FlexLinker while our original version had 2 fluorophores per FlexLinker. This improvement offers increased signal of your FlexAble labeled primaries in fluorescent applications.
Q: Do I need to aliquot the FlexLinker, FlexQuencher and FlexBuffer upon receipt?
A: Aliquoting is optional. FlexAble and FlexAble 2.0 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: FlexAble and FlexAble 2.0 labeling kits are designed to allow you to label as little or as much antibody as you need. You can label as little as 0.5 µg primary antibody with one labeling reaction. The kits can be used to label multiple antibodies with the same kit, if desired.
Q: What is the largest quantity I can label?
A: With a standard kit size (50 reactions), you can label 25 µg of total antibody or up to 50 different antibodies if broken up into reactions. With the 200 reaction size kit, you can label 100 µg of total antibody. You can easily scale up the antibody amount for labeling based on the ratios for one labeling reaction (see table below).
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.
*Note: For every one reaction, there should be 8 µL of total volume. FlexBuffer is used to bring the total volume of the reaction up to 8 µL after adding your primary antibody and 1 µL of FlexLinker together (for one reaction). See the table below for example calculations:
Antibody quantity |
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: How long can I store the FlexAble-labeled antibody?
A: Labeling with FlexAble is fast and easy and requires only a minimal amount of antibody, meaning you can freshly label the exact amount of antibody needed immediately before an experiment. However, if storage of FlexAble-labeled antibody is required, optimal storage conditions should be determined for each individual primary antibody and may vary. Normally, storage of a few days at +4°C is possible.
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 while using the same amount of FlexLinker and FlexQuencher.
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. The kit has also been confirmed to successfully label antibodies in hybridoma supernatant.
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: Because FlexAble and FlexAble 2.0 labeling kits are designed to allow you to label as little or as much antibody as you need, they can be used on multiple occasions. When using our base reaction size (0.5 µg of primary antibody), up to 50 different antibodies can be labeled with our standard size 50 rxn kit. Similarly, up to 10 antibodies can be labeled with our 10 rxn size kit and up to 200 antibodies can be labeled with our 200 rxn size kit if all antibodies are labeled at the one reaction size.
Q: What is the degree of labeling (DOL) achieved by FlexAble Kits?
A: Our original FlexAble Antibody Labeling Kits label each primary antibody with 2 dyes. Our improved FlexAble 2.0 kits label each primary antibody with 5 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 during the labeling process.
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. The same is true for FlexAble 2.0 kits.