Fluorescent-labeled Secondary Antibodies and Streptavidins
Application: Immunofluorescence Staining
The HeLa cells were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100.
Then, the specimen was blocked in BSA/PBS blocking buffer.
Subsequently, the specimen was incubated with properly diluted primary antibody (Mouse Anti α-Tubulin IgG). And, after washing, was further incubated with biotin-conjugated secondary antibody (Goat Anti-Mouse IgG Biotin Conjugate [G0387]).
Finally, it was rinsed with PBS and stained with each fluorescent-probe*.
*Streptavidin FITC Conjugate [S0966] at 5 µg/mL (shown in green), DAPI·2HCl [A2412] at 1 µg/mL (shown in blue)
The HeLa cells were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Then, the specimen was blocked in BSA/PBS blocking buffer.
Subsequently, the specimen was incubated with properly diluted primary antibody (Mouse Anti α-Tubulin IgG and Rabbit Anti KDEL** IgG).
Finally, it was rinsed with PBS and stained with each fluorescent-probe***.
** KDEL (amino acid sequence) normally localize in the endoplasmic reticulum.
*** Goat Anti-Mouse IgG R-PE Conjugate [G0569] at 5 µg/mL (shown in red),
Goat Anti-Rabbit IgG FITC Conjugate [G0452] at 5 µg/mL (shown in green),
Hoechst 33258 [H1343] at 1 µg/mL (shown in blue)
Application: Flow Cytometry
The HeLa cells were incubated with Mouse Anti CD9 Antibody (red line) or Mouse IgG2aκ isotype control (black line) at 5 µg/mL.
Subsequently, both were stained with Goat Anti-Mouse IgG FITC Conjugate [G0406] at 5 µg/mL.
Application: Fluorescent Western Blotting
Transfer of HeLa cell lysate to PVDF membrane. Then, the specimen was blocked in BSA/TBST blocking buffer.
Subsequently, the specimen was incubated with properly diluted primary antibody (Mouse Anti Calnexin IgG and Rabbit Anti α-Tubulin ).
Finally, it was rinsed with TBST and stained with each fluorescent-probe*.
*Goat Anti-Rabbit IgG FITC Conjugate [G0452] at 5 µg/mL(shown in green)
Goat Anti-Mouse IgG R-PE Conjugate [G0569] at 2 µg/mL (shown in red)
- Goat Anti-Mouse IgG FITC Conjugate (green fluorescence)
- Goat Anti-Mouse IgM FITC Conjugate (green fluorescence)
- Goat Anti-Rabbit IgG FITC Conjugate (green fluorescence)
- Streptavidin FITC Conjugate (green fluorescence)
- Goat Anti-Mouse IgG R-PE Conjugate (red fluorescence)
- Goat Anti-Rabbit IgG R-PE Conjugate (red fluorescence)
- Streptavidin R-PE Conjugate (red fluorescence)
- DAPI 2HC (blue fluorescence)
- DAPI 2HCl (0.2mL×5) (1mg/mL in Water) [for Cell Staining] (blue fluorescence)
- Bisbenzimide H 33258 Hydrate (blue fluorescence)
- Bisbenzimide H 33258 (1mg/mL in Water) (0.2mL×5) [for Cell Staining] (blue fluorescence)
Cell Proliferation Assay Reagent
Resazurin, when added to viable cells, is reduced by the cellular enzymatic or chemical reactions converting blue/non-fluorescent resazurin to highly fluorescent resorufin.
The assay is simple to perform since the indicator is water-soluble and has low toxicity, thus eliminating the washing/fixing and extraction steps required in other commonly used cell proliferation assays.
Dead Cell Staining Dye
Acridine orange is a nucleic acid staining dye that is used to identify dead cells. It intercalates with the double-stranded DNA base pairs at a ratio of 1:3 and is capable of emitting green fluorescence (Ex: 500 nm, Em: 520 nm). It also emits red fluorescence (Ex: 460 nm, Em: 650 nm) when bound to RNA or single-stranded DNA. Since acridine orange is mutagenic, this product is supplied as a solution that prevents it from splashing during weighing.
Application: The method of staining cells by A3396
- Remove the medium from the culture plate and wash the cells twice with cold PBS(-). Remove the PBS(-).
- Add PBS(-) and Acridine Orange solution [A3396] (1/50th of the volume of the added PBS(-)) and incubate for 15 minutes.
- Remove the staining solution and wash the cells twice with PBS(-).
- Add PBS(-) and observe the cells under a fluorescence microscope.
Please adjust the staining duration and the volume of the solution according to the cell density.
Some cells may require prior fixation; therefor optimization of the protocol according to your need is recommended.
Chemiluminescence Reagent for the Detection of Superoxide in Mitochondria
Europium Fluorophore DTBTA-Eu3+-labeled Secondary Antibodies and Streptavidin
Figure. SDS-Page of S0993 and G0505 (reducing conditions)
S0993: Detect major band that corresponded to Streptavidin DTBTA-Eu3+.
G0505: Detect 2 major bands that corresponded to the heavy chains and light chains of Goat Anti Mouse IgG DTBTA-Eu3+.
- No cross talk of excitation light
Excitation wavelength λex, max = 335 nm
Emission wavelength λem, max = 616 nm
Sharpened emission spectrum
Large Stokes shift (the difference in wavelength between positions of the band maxima of the absorption and emission spectra)
- Stable fluorescence in various aqueous buffers
Available in Tris, TE, PBS, etc. for wide use
- Long fluorescent life time (τ = 1.02 ms)
Time-resolved fluorometric measurement can remove background fluorescence from the sample matrix and often gives detectability better than one order of magnitude compared to those of conventional fluorometric assays.
Note: Sensitive detection of DTBTA-Eu3+ labeled probes requires time-resolved fluorometric measurements.
Comparison of secondary antibody conjugated to DTBTA-Eu3+ or FITC
Dilute the Mouse IgG to each concentration. Coat 96-well plates with diluted Mouse IgG. Block the plates with BSA/TBST. Incubate with Goat Anti-Mouse IgG Conjugates prepared from DTBTA or FITC at 2.5 µg/mL. After incubation, measure the fluorescence intensity on a plate reader.
DTBTA-Eu3+; excitation=340 nm, emission=620 nm. Lag Time : 450 μsec
FITC; excitation=485 nm, emission=520 nm.
Related Product Category Pages
- Fluorescence-labeled Secondary Antibodies
- Fluorescence-labeled Streptavidins
- Fluorescent Probes
- Europium Fluorescent Labeling Reagent