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|66277S||100 µl (50 tests)||$299.00.0|
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- Analytical tools
SQSTM1/p62 (D5L7G) Mouse mAb (Alexa Fluor® 488 Conjugate) #66277
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Chloroquine #14774 (50 μM, 24 hr; right), using SQSTM1/p62 (D5L7G) Mouse mAb (Alexa Fluor® 488 Conjugate) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).Learn more about how we get our images
Gallery: SQSTM1/p62 (D5L7G) Mouse mAb (Alexa Fluor® 488 Conjugate) #66277
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
- Allow cells to fix for 15 min at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 min each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Block specimen in Blocking Buffer for 60 min.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
posted November 2006
revised November 2013
SQSTM1/p62 (D5L7G) Mouse mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total SQSTM1/p62 protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro220 of human SQSTM1 protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated SQSTM1/p62 (D5L7G) Mouse mAb #88588.
Sequestosome 1 (SQSTM1, p62) is a ubiquitin binding protein involved in cell signaling, oxidative stress, and autophagy (1-4). It was first identified as a protein that binds to the SH2 domain of p56Lck (5) and independently found to interact with PKCζ (6,7). SQSTM1 was subsequently found to interact with ubiquitin, providing a scaffold for several signaling proteins and triggering degradation of proteins through the proteasome or lysosome (8). Interaction between SQSTM1 and TRAF6 leads to the K63-linked polyubiquitination of TRAF6 and subsequent activation of the NF-κB pathway (9). Protein aggregates formed by SQSTM1 can be degraded by the autophagosome (4,10,11). SQSTM1 binds autophagosomal membrane protein LC3/Atg8, bringing SQSTM1-containing protein aggregates to the autophagosome (12). Lysosomal degradation of autophagosomes leads to a decrease in SQSTM1 levels during autophagy; conversely, autophagy inhibitors stabilize SQSTM1 levels. Studies have demonstrated a link between SQSTM1 and oxidative stress. SQSTM1 interacts with KEAP1, which is a cytoplasmic inhibitor of NRF2, a key transcription factor involved in cellular responses to oxidative stress (3). Thus, accumulation of SQSTM1 can lead to an increase in NRF2 activity.
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation. DRAQ5 is a registered trademark of Biostatus Limited. DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries. The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS). This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or firstname.lastname@example.org.