Cells were harvested and lysed in RIPA buffer (EMD Millipore, Billerica, MA, USA) containing Complete Protease Inhibi-tor Cocktail (Hoffman-La Roche Ltd., Basel, Switzerland) plus 1 mM PMSF. Lysates were centrifuged at 12,000 rpm for 20 minutes at 4°C. After quantifications of the protein samples using the BCA Protein Assay Kit (Pierce Biotechonology), total protein was electrophoresed through a 10% SDS–polyacrylamide gel and then transferred to 0.45 lm nitrocellulose membranes (EMD Millipore, CA, USA) on a semi-dry electro transferring unit (Bio-Rad Laboratories Inc.). The following antibodies at indicated dilutions were used for Western blotting: SIRT 1 (1:500 dilution), β-actin antibody; Anti-p53, and phosphorylated p53 antibody (Santa Cruz Biotechnology Inc., 1:1,000 dilution). Protein signals were detected using an ECL detection system (Pierce Biotechonology).
Semi dry electro transferring unit
Manufactured by Bio-Rad
Sourced in United States
The Semi-dry electro transferring unit is a laboratory equipment used for transferring proteins or nucleic acids from a gel to a membrane during the process of Western blotting or Northern blotting. It utilizes a semi-dry method to facilitate the efficient transfer of the target molecules from the gel to the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Tissuelyser system, by Qiagen (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti ho 1 sc 10789, by Santa Cruz Biotechnology (1 mentions)
Anti catalase ab16731, by Abcam (1 mentions)
Horseradish peroxidase linked anti rabbit secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using semi dry electro transferring unit
1
SIRT1 and p53 Western Blotting
2
Epididymal Protein Extraction and Western Blot
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The epididymal tissues (~100 mg) were homogenized in 300 µL of ice-cold protein lysis buffer [50 mM Hepes-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 2.5 mM EGTA (pH 8.0), 1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM Na3VO4, 1 mM DTT, 1% NP-40, 10% glycerol, 0.2 mM PMSF and Protease inhibitor cocktail (Sigma, USA)] using the Tissue Lyser system (Qiagen, USA) with 5 mm sterile stainless steel beads. After centrifugation for 30 min at 10,000 ×g at 4℃, the protein content of the supernatant was measured using a protein assay kit (Bio-Rad, USA). Equal amounts of protein were loaded into the lanes of an SDS-PAGE gel, separated and then transferred to a PVDF membrane using a semi-dry electrotransferring unit (Bio-Rad). After blocking with 5% nonfat milk or bovine serum albumin in TTBS, membranes were probed with specific antibodies diluted in TTBS with 5% nonfat milk or bovine serum albumin as follows: anti-catalase (ab-16731, Abcam, USA), anti-HO-1 (sc-10789, Santa Cruz Biotechnology, USA), anti-p-JNK (s-9251, Cell signaling, USA), anti-70-kDa heat shock cognate protein (HSC70; Santa Cruz Biotechnology) or anti-beta-actin (A5441, Sigma, USA). The membranes were then incubated with an IgG-peroxidase-conjugated secondary antibody for chemiluminescent detection. The band intensities were quantified using Quantity One software (Bio-Rad).
3
BDNF Expression After Traumatic Brain Injury
4
Investigating Protein Signaling Pathways
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Lung tissues were homogenized in a lysis buffer. Protein concentration was determined using a BCA kit. Twenty micrograms of proteins from each sample were subjected to electrophoresis on 12% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes on a semidry electro transferring unit (Bio-Rad, USA). PVDF membranes were blocked with 3% Albumin Bovine V for 2 h and incubated with the diluted primary antibodies against GAPDH(1:4000), calpain1(1:100), calpain2(1:100), pAkt1(1:100), pAkt2(1:100), Akt1(1:100), Akt2(1:100) and PI3K(1:100) overnight at 4 °C. After the overnight incubation with the primary antibody, membranes were washed by PBST (Phosphate Buffered Saline with 0.1% Tween-20) and incubated with HRP-conjugated secondary antibody for 2 h. After extensive washing, blots were detected with enhanced chemiluminescent autoradiography reagent (TIANGEN BIOTECH, China) according to the manufacturer's instruction. GAPDH (37 kD), calpain1 (80 kDa), calpain2 (80 kDa), pAkt1 (60 kDa), pAkt2 (60 kDa), Akt1 (60 kDa), Akt2 (60 kDa) and PI3K (110 kDa) were detected at the indicated approximate molecular sizes. The signal intensity was quantitatively analyzed with Quantity-One analysis software.
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