Cells were washed once in ice-cold 1× PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 50 mM Tris–HCl pH 8.8, 150 mM NaCl) after indicated treatments and cell lysates were boiled at 95 ° C for 5 min. Protein samples were separated on an SDS polyacrylamide gel, transferred onto nitrocellulose membrane (Amersham Protran 0.2 10600001) and blocked with 5% milk in PBS-0.1% Tween. For detection of protein expression the following antibodies were used: IRE1α (Cell Signaling Technology, 3294, 1:1,000), XBP1s (Biolegend, 647502, 1:1,000), FLAG (Sigma-Aldrich, F1804; 1:1,000) and Actin (Sigma-Aldrich, A-5060, 1:5000). Anti-rabbit (111-035-003) and anti-mouse (115-035-003) HRP-conjugated secondary antibodies were purchased from Jackson Immunoresearch and the signal was visualized using Western Blotting Luminol Reagent (SantaCruz, sc-2048).
Anti rabbit 111 035 003 and anti mouse 115 035 003 hrp conjugated secondary antibodies
Manufactured by Jackson ImmunoResearch
Anti-rabbit (111-035-003) and anti-mouse (115-035-003) HRP-conjugated secondary antibodies are used to detect and visualize the presence of primary antibodies that have been raised against rabbit or mouse antigens, respectively. These secondary antibodies are conjugated to the enzyme horseradish peroxidase (HRP), which can be used to catalyze a colorimetric or chemiluminescent reaction for detection purposes.
Automatically generated - may contain errors
Lab products found in correlation
Western blotting luminol reagent, by Santa Cruz Biotechnology (2 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Xbp1s, by BioLegend (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
Protran, by Cytiva (1 mentions)
Ire1a, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
β catenin, by Abcam (1 mentions)
2 protocols using anti rabbit 111 035 003 and anti mouse 115 035 003 hrp conjugated secondary antibodies
1
Western Blot Analysis of UPR Proteins
2
Western Blot Analysis of ER Stress Markers in BMSCs
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BMSCs were washed with cold phosphate‐buffered saline and lysed in 2X sodium dodecyl sulfate (SDS) sample buffer (100 mM Tris‐HCl (pH 6.8), 10 mM EDTA, 4% SDS, and 10% glycine) for 1 h at 4°C, respectively. After centrifugation (12,000g for 15 min), the protein content was measured with an enhanced BCA protein assay kit (Beyotime). Equal amounts (30 μg) of protein in each lane were separated by 10% SDS‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore). For the detection of protein expression, the following antibodies were used: PERK (CST, 3192, 1:1000), ATF6 (Cosmo Bio, AM‐73‐500‐B, 1:1000), TFAR19 (Abcam, ab83958, 1:1000), IGF1 (Cosmo Bio, KM2076, 1:1000), IRE1a (Abcam, ab146176, 1:1000), NRF2 (Abcam, ab62352, 1:1000), β‐catenin (CST 8480, 1:1000). Anti‐rabbit (111‐035‐003), and anti‐mouse (115‐035‐003) HRP‐conjugated secondary antibodies were purchased from Jackson ImmunoResearch and the signal was visualized using western blotting luminol reagent (Santacruz, sc‐2048).
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