Anti-ER stress proteins were analyzed using the ER stress antibody sampler kit (9956, Cell Signaling). Anti-Cellular organelle proteins were assessed using Organelle Maker staining kit (OK7670, ECM Biosciences, Versailles, KY, USA). Goal anti-collagen type 1 antibody was Col-1 (1310-01, SouthernBiotech, Birmingham, AL, USA). Mouse anti-α-tubulin antibody was purchased from cell signaling. Mouse anti-α-actin antibody was purchased Sigma-Aldrich. Rabbit anti-MMP13 antibody was purchased from Novus Biologicals. Non-immune mouse, rabbit, and goat IgGs were purchased from Thermo Fisher Scientific.
Er stress antibody sampler kit
Manufactured by Cell Signaling Technology
The ER stress antibody sampler kit from Cell Signaling Technology contains a selection of antibodies that target proteins involved in the endoplasmic reticulum (ER) stress response. The kit provides a convenient way to detect and study the activation of the ER stress pathway.
Automatically generated - may contain errors
Lab products found in correlation
Col 1, by Southern Biotech (1 mentions)
Mouse anti α tubulin antibody, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Sc 9104, by Santa Cruz Biotechnology (1 mentions)
Mabe343, by Merck Group (1 mentions)
Semi dry transfer apparatus, by Hoefer (1 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Anti vcp, by Abcam (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using er stress antibody sampler kit
1
Comprehensive Protein Analysis Protocol
2
Quantitative Western Blot Analysis of ER Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cell lysates, 20 μg/sample, were run on a 4–12% Bis-Tris polyacrylamide gels using MES as a trailing ion in the buffer (Invitrogen). Proteins were transferred to a nitrocellulose membrane (Whatman) using Towbin buffer with 20% (v/v) methanol in a semi-dry transfer apparatus (Hoefer) and reversibly stained with Ponceau S for loading control. Membranes were incubated in TBS buffer containing 0.01% Tween-20 and 5% (w/v) skim milk powder (Fluka), containing the primary antibodies. The following antibodies were used: ER stress antibody sampler kit (all 1:1000; #9956, Cell Signaling Technology; including secondary antibodies used here), anti-MAFF (1:500; AV38984, Sigma-Aldrich), anti-eIF4G1 (1:1000; 2858S, Cell Signaling Technology), anti-tubulin (1:1000; sc-9104, Santa Cruz Biotechnology). Puromycin incorporation was detected using the 12D5 mouse monoclonal antibody (MABE343, Millipore) at a 1:5000 final concentration. The quantification of gel bands was performed using the image processing program ImageJ (http://imagej.nih.gov/ij ) (Schneider et al., 2012 (link)).
3
Subcellular Fractionation and Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected in 1% NP-40 containing 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl2. After centrifugation at 15,000 × g for 5 min, the supernatant was collected and analyzed as the soluble fraction. The pellet was re-suspended in 1% SDS containing 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl2 and analyzed as the insoluble fraction. Those fractions were boiled for 5 min in SDS–PAGE sample buffer containing 0.125 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, and 0.004% bromophenol blue and loaded onto a 5–20% or 10–20% polyacrylamide gradient gel. The ER stress antibody sampler kit was obtained from Cell Signaling Technology. Blue native-PAGE was performed as previously described (Bin et al, 2011 (link)). Anti-V5 (Invitrogen), anti-tubulin (Santa Cruz), anti-ubiquitinated proteins (Biomol), anti-FLAG (Sigma), and anti-VCP (Abcam) antibodies, and an anti-ER stress antibody sampler kit (Cell Signaling) were used for protein detection.
4
Antibody Detection of Enteroviruses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect CVA9, CVB1, and CVB3, either polyclonal rabbit antiserum against CVA and CVB (kindly provided by Merja Roivainen, National institute of Health and Welfare, Helsinki, Finland) or monoclonal antibody against EVs (ncl-entero; clone 5-D8/1) (Novocastra) was used. For detection of EV1, rabbit antiserum against purified EV1 (66 (link)) was used. Antibodies against the ER stress markers were obtained from the ER stress antibody sampler kit (Cell Signaling Technologies). Other antibodies were monoclonal (NCL-VIM-V9; Leica Microsystems) and rabbit polyclonal antibody against vimentin (H-84) (Santa Cruz Biotechnology, Inc.), in addition to monoclonal antibody against dsRNA (J2; English & Scientific Consulting). GM130 and PDI antibodies were from Abcam, and G3BP1, PABP, eIF4G, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were from Santa Cruz. Antibody against beta-tubulin was from Cedarlane. Viral protease antibodies have been previously published (67 (link)). Antibody against 3D was a kind gift from Antonio Toniolo (Università dell’Insubria, Italy).
5
Neuroblastoma and Leukemia Cell Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CHLA255 (neuroblastoma), SubB15 (acute lymphoblastic leukemia, ATCC CRL-1929) and NALM6 (a B cell precursor leukemia cell line, ATCC CRL-3273) cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin until log phase. Cell lysates of neuroblastoma (CHLA255), leukaemia cells (SupB15 or NAML-6) were treated with different amounts of NK-EVs as indicated in the figure legend. For each treatment, 30 µg of cell lysate proteins were loaded per lane and separated by SDS-PAGE, and proteins were electro-transferred to PVDF membranes (Millipore). The blots were probed with antibodies to HMG2 (Cell Signalling Technology, #14163, 1:500) or SET (Santa Cruz Biotech, sc-133138, 1:500) followed by incubation with anti-mouse-HRP secondary antibody conjugates (1:5,000 dilution). Proteins were detected with a chemiluminescence substrate (cat. #34080) from ThermoFisher Scientific, Inc. (Waltham, MA). ER Stress Antibody Sampler Kit was purchased from Cell Signalling Technology (#9956S). Protein size markers were purchased from Bioland Scientific, LLC. (cat. # PM01-01).
6
Investigating ER Stress in Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ER stress was investigated using the ER stress Antibody Sampler Kit
(Cell Signaling, Danvers, MA). Fibroblasts grown under normal cell culture
conditions as described above were used and additionally, cells stressed by
glucose starvation were studied. To starve the cells, Dulbecco’s modified
Eagle’s medium (25 mM glucose) was removed; the fibroblasts were washed with
phosphate buffered saline and then incubated for 12 h in Dulbecco’s modified
Eagle’s medium with glucose concentration reduced to 0.5 mM glucose, 10% fetal
bovine serum, and 1% penicillin/streptomycin at 37 °C and 5%
CO2.
(Cell Signaling, Danvers, MA). Fibroblasts grown under normal cell culture
conditions as described above were used and additionally, cells stressed by
glucose starvation were studied. To starve the cells, Dulbecco’s modified
Eagle’s medium (25 mM glucose) was removed; the fibroblasts were washed with
phosphate buffered saline and then incubated for 12 h in Dulbecco’s modified
Eagle’s medium with glucose concentration reduced to 0.5 mM glucose, 10% fetal
bovine serum, and 1% penicillin/streptomycin at 37 °C and 5%
CO2.
7
ER Stress Induction via Glucose Starvation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!