Products

KyoBright488-conjugated Secondary Antibody

This product is a fluorescently conjugated secondary antibody that can be used for applications such as immunocytochemistry. Two affinity-purified antibody types are available: Anti-Mouse IgG and Anti-Rabbit IgG. KyoBright488, the fluorophore conjugated to this product, is a bright and highly photostable fluorescent dye. Like the widely used FITC and Alexa Fluor 488, it is excited by blue light and emits green fluorescence.

Features

  • Detectable with a standard 488 nm filter set (Ex 495 nm / Em 520 nm)
  • Affinity purified
  • Cross-adsorbed*

    *Anti-Mouse IgG: cross-adsorbed against Human IgG, Rat IgG, and Rabbit IgG
    *Anti-Rabbit IgG: cross-adsorbed against Human IgG, Rat IgG, and Mouse IgG

Product Description

Performance comparison with competitor products in immunocytochemistry staining

Fluorescence microscopy observation

MAP2 or GFAP was detected using primary cultured mouse neurons and primary cultured mouse astrocytes. This product showed results comparable to those of Company A's product.

Primary_cultured.png

<Experimental conditions>
Sample : [MAP2] Mouse primary cultured neurons
[GFAP] Mouse primary cultured astrocytes
Fixation : 2% paraformaldehyde phosphate buffer solution, room temperature, 30 min
Permeabilization : 0.1% Triton X-100 in PBS, room temperature, 10 min
Blocking : Blocking One Histo (#06349-64), room temperature, 15 min
Primary antibody : [MAP2] MAP2 antibody (Sigma-Aldrich #M4403), 1:1000 dilution, overnight at 4°C
[GFAP] GFAP antibody (Invitrogen #PA1-10019), 1:1000 dilution, overnight at 4°C
Antibodies were diluted 20-fold with Blocking One Histo / PBS
Secondary antibody / staining reagent : [MAP2] KyoBright488-conjugated secondary antibody (#22844) or competitor fluorescent secondary antibody, 1:500 dilution (4 μg/mL), room temperature, 1 h
[GFAP] KyoBright488-conjugated secondary antibody (#22849) or competitor fluorescent secondary antibody, 1:500 dilution (4 μg/mL), room temperature, 1 h
[DAPI] DAPI solution (1 mg/mL) (#19178-91), 1:1000 dilution
Antibodies were diluted 20-fold with Blocking One Histo / PBS
Observation : Fluorescence microscope

Detection by flow cytometry

Expression of Nestin or Sox2 was confirmed using mouse neural stem cells. This product showed results comparable to those of Company A's product.

Detection_by_flow_cytometry.png
<Experimental conditions>
Sample : Neural stem cells isolated from mouse embryos (E9.5)
Fixation : 1% paraformaldehyde phosphate buffer solution, room temperature, 20 min
Permeabilization : 0.1% Triton X-100 in PBS, 4°C, 15 min
Primary antibody : [Nestin] Nestin antibody (Invitrogen #MA1-110), 1:100 dilution, overnight at 4°C
[SOX2] SOX2 antibody (Invitrogen #PA1-094), 1:100 dilution, overnight at 4°C
Secondary antibody : [Nestin] KyoBright488-conjugated secondary antibody (#22844) or competitor fluorescent secondary antibody, 1:200 dilution (10 μg/mL), room temperature, 1 h
[SOX2] KyoBright488-conjugated secondary antibody (#22849) or competitor fluorescent secondary antibody, 1:200 dilution (10 μg/mL), room temperature, 1 h
Detection : Flow cytometer — Attune NxT Acoustic Focusing Cytometer (Invitrogen)

Evaluation of fluorophore stability

Using immunocytochemistry staining with HEK293 cells, the stability of the fluorophore under excitation light exposure and longterm storage was evaluated. This product was shown to exhibit dye stability equivalent to that of Company A's product.


Comparison of dye stability under excitation light exposure

Comparison_of_dye_stability_under_excitation_light_exposure.png

Comparison of dye stability after long-term storage

Comparison_of_dye_stability_after_longterm_storage.png
<Experimental conditions>
Sample : HEK293 cells
Fixation : 2% paraformaldehyde phosphate buffer solution, room temperature, 30 min
Permeabilization : 0.1% Triton X-100 in PBS, room temperature, 10 min
Blocking : Blocking One Histo (#06349-64), room temperature, 15 min
Primary antibody : β-Actin antibody (Santa Cruz #sc-47778), 1:150 dilution, overnight at 4°C
Antibodies were diluted 20-fold with Blocking One Histo / PBS
Secondary antibody : [FITC] FITC-conjugated secondary antibody, 1:50 dilution (12 μg/mL)
[This product] KyoBright488-conjugated secondary antibody (#22844), 1:500 dilution (4 μg/mL)
[Company A] Company A's fluorescent secondary antibody, 1:500 dilution (4 μg/mL)
Antibodies were diluted 20-fold with Blocking One Histo / PBS and reacted at room temperature for 2 h
Mounting medium : [Fixation compatible] 85% glycerin-containing PBS
[Long-term storage] Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (#12745)
Observation : Fluorescence microscope

Application

Application to fluorescent Western blotting

Using HEK293 cells treated to activate Protein Kinase C and iPS cell-derived hepatocytes, multiplex detection was performed for housekeeping proteins as well as proteins involved in phosphorylation and differentiation.

Application_to_fluorescent_Western_blotting.png

Green: KyoBright488-conjugated secondary antibody
Blue: StarBright Blue 700-conjugated secondary antibody

<Experimental conditions>
Sample : ① HEK293 cells (10 μg)
② HEK293 cells treated for Protein Kinase C activation (10 μg)
③ iPS cells (10 μg)
④ iPS cell-derived hepatocytes (10 μg)
SDS-PAGE : Bullet PAGE Plus Precast Gel, 5–20%, 17 wells (#21794-54), 400 V
Transfer : Bullet Semi-dry Transfer One (#15353), 25 V, 10 min
Immun-Blot Low Fluorescence PVDF (Bio-Rad)
Blocking : Bullet Blocking One for Western Blotting (#13779), room temperature, 5 min
Primary antibody : [β-Actin] β-Actin antibody (Santa Cruz #sc-47778), 1:500 dilution
[pERK] pERK antibody (CST #4370S), 1:1000 dilution
[GAPDH] GAPDH antibody (Novus Biologicals #NB300-322), 1:1000 dilution
[αAT] αAT antibody (Proteintech #66135-1-Ig), 1:5000 dilution
Antibodies were diluted with 0.1% TBS and reacted at room temperature for 1 h
Secondary antibody : [β-Actin] KyoBright488-conjugated secondary antibody (#22844), 1:2500 dilution
[pERK] StarBright Blue 700-conjugated secondary antibody (Bio-Rad #12004161), 1:2500 dilution
[GAPDH] KyoBright488-conjugated secondary antibody (#22849), 1:2500 dilution
[αAT] StarBright Blue 700-conjugated secondary antibody (Bio-Rad #12004158), 1:2500 dilution
Antibodies were diluted with 0.1% TBS and reacted at room temperature for 1 h
Detection : ChemiDoc Touch MP (Bio-Rad)
[β-Actin, GAPDH] 532 / 28 nm filter (Blue excitation)
[pERK, αAT] 715 / 30 nm filter (Blue excitation)

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Ordering Information

Product name Grade Storage Catalog number PKG size Price
Anti-Mouse IgG(Goat), KyoBright488-conjugated, Pre-absorbed SP Refrigerate 22844-06 100 μL e-Nacalai
22844-64 0.5 mL e-Nacalai
Anti-Rabbit IgG(Goat), KyoBright488-conjugated, Pre-absorbed SP Refrigerate 22849-56 100 μL e-Nacalai
22849-14 0.5 mL e-Nacalai

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