Cells were washed once with PBS, suspended in PBS (100 μL/106 cells), and mixed with an equal volume of 2× sample buffer. Samples were heated at 95°C with occasional vortexing until samples were completely dissolved. Samples were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and reacted with primary antibodies as described in the text. Human MRC1 was identified using a rabbit monoclonal antibody to hMRC1 (clone ab125028; Abcam, Cambridge, MA, USA). HIV-1 Gag was identified using pooled HIV immunoglobulin (Ig; NIH Research and Reference Reagent Program; Cat no. 3957), and tubulin was identified using a mouse monoclonal antibody to alpha-tubulin (T9026; Sigma-Aldrich, St. Louis, MO, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA), and proteins were visualized by enhanced chemiluminescence (Clarity Western ECL substrate 170-5061; Bio-Rad Laboratories, Hercules, CA, USA).
Clarity western ecl substrate 170 5061
Manufactured by Bio-Rad
Sourced in United States
Clarity Western ECL substrate 170-5061 is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It provides a sensitive and reliable method for visualizing target proteins on a nitrocellulose or PVDF membrane.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase linked secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (3 mentions)
Ab125028, by Abcam (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (2 mentions)
T9026, by Merck Group (2 mentions)
Vinculin ma5 11690, by Merck Group (2 mentions)
Herc1 a301 904a, by Fortis Life Sciences (2 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
C abl sc 23, by Santa Cruz Biotechnology (1 mentions)
Criteriontm tgx stain freetm, by Bio-Rad (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Anti tubulin t9026, by Merck Group (1 mentions)
Anti gm130 ab52649, by Abcam (1 mentions)
Hrp conjugated goat anti mouse igg 31 430, by Thermo Fisher Scientific (1 mentions)
Anti sec31a, by BD (1 mentions)
Dc protein assay 500 0119, by Bio-Rad (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
6 protocols using clarity western ecl substrate 170 5061
1
Western Blot Analysis of MRC1 and HIV-1 Gag
2
Western Blot Protein Analysis Protocol
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Seventy µg of total proteins were loaded and run onto 10% Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred to PVDF (Bio-Rad, Hercules, California, CA, USA) membranes. Membranes were blocked in TBS (Tris-HCl pH 7.4, 150 mM NaCl) plus 5% BSA for 1 h at room temperature (RT) and then decorated with appropriate antibodies (Sprouty1 sc-365520, Tubulin sc-23948, and phospho-Akt1/2/3 (Thr308) sc-16646, Santa Cruz Biotechnology; TATA Binding protein (TBP) MA1-189 and Vinculin MA5-11690, Sigma-Aldrich; Foxo3a #2497, p44/42 MAPK (ERK1/2) #4696s, phospho-p44/42 MAPK (T202/Y204) #4377s and Akt (pan) (C67E7) #4691s, Cell Signaling) in PBS-Tween 0.2% overnight at 4 °C. Membranes were then washed with PBS-Tween 0.2% three times for 15 min each, incubated with appropriate peroxidase-linked secondary antibody (Santa Cruz Biotechnology) for 1 h at RT and washed again in PBS-Tween 0.2%. Specific binding was detected using an enhanced chemiluminescence system (Clarity Western ECL Substrate #170-5061, Bio-Rad).
3
Western Blot Analysis of Protein Expression
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Protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad). 100 µg of total protein were loaded and run onto 4–14% precast gradient gel (Criterion TM TGX Stain-Free TM) electrophoresis and transferred to PVDF (Bio-Rad, Hercules, California, CA, USA) membranes by using the protocol described elsewhere [22 (link)]. Blots were blocked in TBS (Tris-HCl pH7.6, 150 mM NaCl) plus 5% nonfat milk for 1 h at room temperature (RT) and then decorated with appropriate antibodies (HERC1 A301-904A, Bethyl Laboratories, Inc., Montgomery, TX, USA, Tubulin sc-23948, and p44/42 MAPK (ERK1/2) #4696s, phospho-p44/42 MAPK (T202/Y204) #4377s; Vinculin MA5-11690, Sigma-Aldrich; BCR Cell Signaling Tech, phospho-Tyr, sc-7020, Santa Cruz Biotechnology, Dallas, TX, USA; in PBS-Tween 0.5% overnight at 4 °C. Membranes were then washed with PBS-Tween 0.5% 3 times for 15 min each, incubated with appropriate peroxidase-linked secondary antibody (Santa Cruz Biotechnology) for 1 h at RT, and washed again in PBS-Tween 0.5%. Specific binding was detected using an enhanced chemiluminescence system (Clarity Western ECLSubstrate #170-5061, Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Protein Samples
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One hundred micrograms (100 µg) of total protein were loaded and run onto 4%–14% gradient precasted gel (CriterionTM TGX Stain-FreeTM, Bio-Rad, Hercules, CA, USA) electrophoresis and transferred to PVDF (Bio-Rad, Hercules, CA, USA) membranes as described elsewhere [42 (link)]. Blots were blocked in TBS plus 5% non-fatty acid milk and decorated with appropriate primary antibodies (HERC1 #A301-904A, Bethyl Laboratories Inc., Montgomery, TX, USA; β-Actin, sc-47778, phospho-Tyr, sc-7020, c-Abl, sc-23 and BCR, sc-20707, Santa Cruz Biotechnology, Dallas, Texas, USA; Vinculin MA5-11690, Invitrogen, Carlsbad, CA, USA). Membranes were then washed and incubated with appropriate peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Specific binding was detected using an enhanced chemiluminescence system (Clarity Western ECL Substrate #170-5061, Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of MRC1 and HIV-1 Gag
6
Comprehensive Reagents and Antibody Protocol
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Reagents and antibodies were purchased from the following suppliers: HRP–conjugated goat anti-mouse IgG (31430) and goat anti-rabbit IgG (31461); Alex Fluor 488 goat anti-mouse IgG (A11029), Texas Red goat anti-mouse IgG (T-862), and goat anti-rabbit IgG (T-2767); and Alexa Fluor Plus 647 goat anti-mouse IgG (A32728) and goat anti-rabbit IgG (A32733) are from Thermo Fisher Scientific. Anti-beclin-1 (NB500-249), anti-FAM134B (NBP2-55248), and anti-Sec62 (NBP1-84045) are from Novus Biologicals. Anti-calnexin (C4731), anti-HA (H9658), anti-LC3B (L7543), anti-Myc (C3956), and anti-tubulin (T-9026) are from Sigma-Aldrich. Anti-ERGIC53 (ALX-804-602) is from Enzo Life Sciences. Anti-GABARAP+L1+L2 (ab109364) and anti-GM130 (ab52649) are from Abcam. Anti-LAMP2 (555803) and anti-Sec31A (612351) are from BD Pharmingen. Anti-p62 (H00008878-M0162) is from Abnova. Anti-VMP1 (12978s) is from Cell Signaling. DC Protein Assay (500-0119) and Clarity Western ECL Substrate (170-5061) are from Bio-Rad. Effectene Transfection Reagent (301427) is from Qiagen. Lipofectamine RNAiMAX Reagent (100014472) is from Thermo Fisher Scientific. MTS cross-linking reagents M3M (1,3-propanediyl bis-MTS) and M8M (1,5-pentanediyl bis-MTS) are from Toronto Research Chemicals. ProLong Diamond antifade mountant without (P36970) or with DAPI (P36971) is from Thermo Fisher Scientific.
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