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Published TCIMAIL newest issue No.198
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Fluorescent-labeled Secondary Antibodies and Streptavidins
Application: Immunofluorescence Staining
(Laser Scanning Microscope: Olympus FLUOVIEW FV 3000)
Green: α-Tubulin，Blue: Nuclear DNA
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)
(Laser Scanning Microscope: Olympus FLUOVIEW FV 3000)
Red: α-Tubulin, Green: Endoplasmic Reticulum, Blue: Nuclear DNA
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
(Flow cytometer: Sysmex RF-500)
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
(Imager: Cytiva Amersham Imager 680)
Red: Calnexin, Green: α-Tubulin
Lane 1: Color Protein Marker, Lane 2: HeLa Whole Cell Lysate at 5 µg
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)
Products
- G0406
- Goat Anti-Mouse IgG FITC Conjugate (green fluorescence)
- G0453
- Goat Anti-Mouse IgM FITC Conjugate (green fluorescence)
- G0452
- Goat Anti-Rabbit IgG FITC Conjugate (green fluorescence)
- S0966
- Streptavidin FITC Conjugate (green fluorescence)
- G0569
- Goat Anti-Mouse IgG R-PE Conjugate (red fluorescence)
- G0577
- Goat Anti-Rabbit IgG R-PE Conjugate (red fluorescence)
- T3885
- Streptavidin R-PE Conjugate (red fluorescence)
- A2412
- DAPI 2HC (blue fluorescence)
- D5888
- DAPI 2HCl (0.2mL×5) (1mg/mL in Water) [for Cell Staining] (blue fluorescence)
- H1343
- Bisbenzimide H 33258 Hydrate (blue fluorescence)
- B6236
- Bisbenzimide H 33258 (1mg/mL in Water) (0.2mL×5) [for Cell Staining] (blue fluorescence)
Cell Proliferation Assay Reagent
Resazurin solution [R0195] can be used quantitatively determine cell proliferation, viability, and cytotoxicity.
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.
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.
Product
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.
Product
Application: The method of staining cells by A3396
NIH3T3 cells stained 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.
λDNA was stained using either the acridine orange solution [A3396] or the other manufacturer's product, and the fluorescence measured from two experiments was compared (Ex:500nm, Em:520nm).
The results indicated that A3396 stained better than the other manufacturer's product.
Chemiluminescence Reagent for the Detection of Superoxide in Mitochondria
Figure. Fluorescence in mitochondria in mouse peritoneal neutrophils. (Provided by Prof. Kobayashi)
S. Sasaki, S. Yamada, M. Iwamura, Y. Kobayashi, Free Radic. Biol. Med. 2013, 65, 1005.
MMT [B4339] is a specific probe having lucigenin-like chemiluminescence to superoxide among reactive oxygen species. Since amphiphilic MMT which is less hydrophilic than lucigenin possesses cell-permeability, MMT has been applicable for the detection of intramitochondrial superoxide production.
Product
Europium Fluorophore DTBTA-Eu3+-labeled Secondary Antibodies and Streptavidin
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+.
DTBTA-Eu3+ Advantages
- 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
[Assay condition]
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.
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.