Protein samples from total cell extracts were washed with ice-cold phosphate-buffered solution (PBS). Protein samples were lysed in Pro-Prep buffer containing a phosphatase inhibitor cocktail solution (iNtRON Biotechnology, Seongnam-si, Korea) for 30 min on ice followed by centrifugation at 13,000× g for 20 min at 4 °C. Equal amounts of protein from each supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA). Then the blots were probed overnight at 4 °C with monoclonal antibodies against Islet 1 (Cat. no. ab86472), HB9 (Cat. no. ab79541), or ChAT (Cat. no. ab178850) (1:500, all from Abcam, Cambridge, UK) followed by the corresponding secondary antibody. The blots were developed using enhanced chemiluminescence reagents (WestSave Gold Western Blot Detection kits; AbFrontier, Seoul, Korea). The intensity of each band was assessed by densitometric scanning (LAS-3000; Fujifilm, Tokyo, Japan). The expression level of each protein was normalized with the respect of GAPDH levels (No. LF-PA0018; 1:1000, pRb, AbFrontier). The protein levels were quantified using Multi Gauge software (version 3.0; Fuji Photo Film, Kanagawa, Japan).
Lf pa0018
Manufactured by AbFrontier
The LF-PA0018 is a laboratory equipment product. It is a precision device used for a specific function in laboratory settings. No further details about its core function or intended use are available.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene membrane, by Merck Group (2 mentions)
Westsave gold western blot detection kits, by AbFrontier (2 mentions)
Phosphatase inhibitor cocktail solution, by iNtRON Biotechnology (2 mentions)
Las 3000, by Fujifilm (2 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Nucleospin rna protein, by Macherey-Nagel (1 mentions)
Z0334, by Fujifilm (1 mentions)
Nb100 1028, by Santa Cruz Biotechnology (1 mentions)
Lf qc0101, by AbFrontier (1 mentions)
Ab59225, by Abcam (1 mentions)
Ab117840, by Abcam (1 mentions)
Ab32678, by Abcam (1 mentions)
Sc 7947, by Santa Cruz Biotechnology (1 mentions)
Prmt1, by Merck Group (1 mentions)
α actinin, by Merck Group (1 mentions)
Sc 8784, by Santa Cruz Biotechnology (1 mentions)
N cadherin antibody, by BD (1 mentions)
Pa5 29559, by Thermo Fisher Scientific (1 mentions)
Zonula occludens 1, by Thermo Fisher Scientific (1 mentions)
Ab5694, by Abcam (1 mentions)
4 protocols using lf pa0018
1
Western Blot Analysis of Neuronal Markers
2
Brain Tissue Isolation and Protein Analysis
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For brain tissue preparation, mice were deeply anesthetized with Zoletil (12.5 mg/kg) and Rompun mix (17.5 mg/kg) administered intraperitoneally. Mice were perfused transcardially with heparin dissolved in PBS (pH 7.2). The dissected brain tissues were frozen at -80 °C for Western blotting. Tissues were homogenized with total RNA and protein isolation kit (NucleoSpin® RNA/Protein #740933.50, Macherey-Nagel, Dűren, Germany). The protein samples were quantified with Pierce™ BCA Protein Assay Kit and 50 μg protein sample were used for each Western blot. The primary antibodies were applied in the following concentrations: anti-GFAP (rabbit, 1: 1,000; Dako #Z0334), anti-Iba1 (rabbit, 1:300; Wako #NB100-1028), anti-EAAT1 (rabbit, 1:1,000; Santacruz # sc-15316), anti-EAAT2 (rabbit, 1:1,000; Cellsignaling #3838), anti-Cx43 (mouse, 1:1,000; Santacruz #sc-59949), anti-PSD95 (mouse, 1:2,000; Thermo #MA1-046), anti-NeuN (mouse, 1:1,000; Millipore #MAB377) anti-GAPDH (rabbit, 1:10,000; Abfrontier #LF-PA0018). Secondary antibodies were conjugated with horse-radish peroxidase (HRP) (1: 10,000, Invitrogen). The HRP signals were visualized using an enhanced chemiluminescent (ECL, Abfrontier #LF-QC0101, Gyeonggi-do, Korea) substrate.
3
Comprehensive Antibody Characterization
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Antibodies against p-CaMKII (1:500, ab32678), p-RyR2(S2808) (1:500, ab59225), RyR2 (1:200. ab117840), α-SMA (1:1000, ab5694), and Desmoplakin (1:200, ab106342) were purchased from Abcam. Flag-M2 (1:1000, F1804) and α-Actinin (1:1000, A7737) were obtained from Sigma-Aldrich. Antibodies against Gapdh (1:1000, LFPA0018) and HA (1:1000, LFMA0048) were from AbFrontier. Collagen1 (1:1000, SC-8784) and HSP90 (1:1000, SC-7947) were obtained from Santa Cruz Biotechnology. PRMT1 (1:500, 07-404) and Oxi-CaMKII (1:500, 07-1387) were from Millipore. N-cadherin antibody (1:500, 610921) was from BD biosciences. ANP (1:500, PA5-29559) and Zonula Occludens-1 (1:200, 40-2200) were from Thermo Fisher Scientific. Antibodies against ASYM (1:200, 13522), Connexin43 (1:1000, 3512) and CaMKII (1:1000, 3362) were from Cell Signaling Technology. Anti-mouse (1:5000, 115-035-033), anti-rabbit (1:5000, 111-035-003) IgG peroxidase-conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories and anti-goat (1:5000, 81-1620) IgG peroxidase-conjugated secondary antibodies was ordered from Thermo Fisher Scientifics.
4
Akt and ERK Protein Expression Analysis
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Sciatic nerve tissues were washed with ice-cold PBS and lysed in Pro-Prep buffer containing a phosphatase inhibitor cocktail solution (iNtRON Biotechnology, Seongnam-si, Korea) for 30 min on ice. After centrifugation at 13,000× g for 20 min at 4 °C, equal quantities of protein from supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and were electrophoretically transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA). The blots were then probed overnight at 4 °C with antibody against v-Akt Murine Thymoma Viral Oncogene (Akt; No. 9272) or Phospho-Akt (p-Akt; Thr308; No. 9275) or extracellular-signal-regulated kinase (ERK; Phospho-p44/43 MAPK (Erk1/2) (Thr202/Tyr204); No. 9101) (1:500, all pRb, Cell signaling) followed by the corresponding secondary antibody. The blots were washed and developed using enhanced chemiluminescence reagents (WestSave Gold Western Blot Detection kits; AbFrontier, Seoul, Korea), according to the manufacturer’s instructions. Band intensities were assessed by densitometric scanning (LAS-3000, Fujifilm, Tokyo, Japan). The p-Erk protein expression level was normalized to the expression of GAPDH (No. LF-PA0018; 1:1000, pRb, Ab Frontier, Korea). The results were quantified using Multi Gauge software, version 3.0.
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