Figure 1. SQSTM1/p62 protein from SK-MEL-2 cells is detected using PathScan® Total SQSTM1/p62 Sandwich ELISA Kit #7814. This kit detects lower levels of SQSTM1/p62 in SK-MEL-2 cells starved overnight in Earle's Balanced Salt Solution (EBSS). The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using SQSTM1/p62 (D5E2) Rabbit mAb #8025 is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from unstarved and EBSS-starved SK-MEL-2 cells and the absorbance at 450 nm is shown. SK-MEL-2 cells (85% confluence) were starved overnight in EBSS at 37ºC and then lysed.
|REACTIVITY||H M R Mk Mi|
|Product Includes||Volume||Solution Color|
|SQSTM1/p62 Rabbit mAb Coated Microwells||96 tests|
|SQSTM1/p62 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total SQSTM1/p62 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total SQSTM1/p62 protein. An SQSTM1/p62 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, SQSTM1/p62 protein is captured by the coated antibody. Following extensive washing, SQSTM1/p62 Mouse Detection Antibody is added to detect the captured SQSTM1/p62 protein. Anti-Mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of total SQSTM1/p62 protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total SQSTM1/p62 Sandwich ELISA Kit detects endogenous levels of SQSTM1/p62 protein in human cells as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
Human, Mouse, Rat, Monkey, Mink
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.
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PathScan is a trademark of Cell Signaling Technology, Inc.