REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
M R | Endogenous | 62 | Rabbit IgG |
Western blot analysis of extracts from MEFs from wild-type or SQSTM1/p62 knockout mice using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.
Learn more about how we get our images.Western blot analysis of extracts from C2C12 cells, untreated (-) or treated with Chloroquine #14774 (50 μM, overnight) using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Learn more about how we get our images.Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).
Learn more about how we get our images.Western blot analysis of extracts from MEFs, untreated (-) or treated with Earles Basic Salt Solution (EBSS; 4 hr; +) using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Learn more about how we get our images.Immunoprecipitation of SQSTM1 from L-929 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific). Western blot was performed using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific). Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded MEF wild-type cell pellet (left, positive) or MEF SQSTM1/p62 KO cell pellet (right, negative) using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded mouse forestomach using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded mouse kidney using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded mouse spleen using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded rat spleen using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded mouse small intestine using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).
Learn more about how we get our images.Confocal immunofluorescent analysis of wild-type MEFs, either untreated (left) or treated with Chloroquine #14774 (50 μM, 18 hours; center), and SQSTM1/p62 knock-out MEFs treated with chloroquine (right), using SQSTM1 (D6M5X) Rabbit mAb (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.
Learn more about how we get our images.For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 409
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 283
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
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.
posted November 2006
revised December 2010
Protocol Id: 32
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
Immunohistochemistry (Paraffin) | 1:250 |
Immunofluorescence (Immunocytochemistry) | 1:800 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) recognizes endogenous levels of total rodent SQSTM1/p62 protein.
Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly300 of mouse SQSTM1/p62 protein.
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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SignalStain is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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Product # | Size | Price |
---|---|---|
23214S | 100 µl | N/A |
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