Adherent cells were lysed in NP-40 buffer containing protease cocktail inhibitor (Roche cat#11-836-170-001), sonicated, and centrifuged at 14,000rpm for 15 min at 4°C. Lysates were collected and protein concentration was determined using the BCA assay (Thermo cat#23225). 35–50μg of protein was prepared in NuPAGE sample buffer (Invitrogen cat#NP0007) and reducing agent (Ivitrogen cat#NP0004). Proteins were separated on a SDS-PAGE gel under reducing conditions and were transferred to nitrocellulose membranes (Bio-Rad cat#1620115). The membrane was then blocked in 1% fish gelatin blocking buffer (Amresco cat#M319) at room temperature for 1 hour and primary antibodies (1:1000 Myc-tag (71D10) rabbit monoclonal antibody, Cell Signaling Technology cat#2278; 1:1000 FLAG-tag rabbit polgyclonal antibody, Invitrogen cat#PA1-984B) were applied overnight at 4°C. GAPDH (Sigma cat#G9295) loading control was probed on membranes at 1:20,000 for 1 hour room temperature. SuperSignal West Pico Chemiluminescent Substrate (Thermo cat#34077) was used for detection.
Fish gelatin blocking buffer
Manufactured by Avantor
Sourced in United States
Fish gelatin blocking buffer is a buffer solution designed for use in various laboratory techniques, such as immunoassays and Western blotting. The primary function of this buffer is to prevent non-specific binding of antibodies or other proteins to the test surface, thereby reducing background signals and improving the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Infinite 200, by Tecan (3 mentions)
Las4000, by GE Healthcare (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Erk 1 2 and phospho erk 1 2 primary antibodies, by Cell Signaling Technology (1 mentions)
Anti cxcr3 antibody, by Abcam (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using fish gelatin blocking buffer
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Western Blot Analysis
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Cells were washed with PBS and lysed by M-PER mammalian protein extraction reagent (Thermo, IL, USA) with Halt protease inhibitor cocktail (Thermo, IL, USA) and were then centrifuged at 14,000g for 10 min at 4°C to collect the precleared cell extracts. Protein concentration was determined with the Coomassie Plus (Bradford) protein assay reagent (Thermo, IL, USA) using multimode microplate readers (Infinite 200, Tecan). Protein samples were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany). Membranes were blocked in 10% fish gelatin blocking buffer (Amresco, OH, USA) for 1 hour and then incubated with the anti-human active form of PAR1 primary antibody (Sigma, MO, USA) at 1 : 2500 dilution and ERK 1/2 and phospho-ERK 1/2 primary antibodies (Cell Signaling Technology, MA, USA) at 1 : 2500 dilution at 4°C overnight. The blots were washed with Tris-buffered saline with Tween 20 (TBST) and incubated with goat anti-rabbit secondary antibody for 1 hour at room temperature. Membranes were washed and then detected by an enhanced chemiluminescence substrate using a luminescence imaging system (LAS 4000, GE, USA).
3
Fractionation and Detection of CXCR3 Protein
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Cells were washed with 1× PBS before the cytosolic and membrane proteins were fractionated by Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo, IL, USA), according to the manufacturer’s instructions. Protein concentration was determined with the Coomassie Plus (Bradford) protein assay reagent (Thermo, IL, USA) using multimode microplate readers (Infinite 200, TECAN). Protein samples were resolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany). Membrane was blocked in 10% fish gelatin blocking buffer (AMRESCO, OH, USA) for 1 h and then incubated with the primary anti-CXCR3 antibody (Abcam, London, UK) at 1:3000 dilution at 4 °C overnight. Pan-cadherin (GeneTex, CA, USA) was used as a cell plasma membrane marker. The blots were washed with Tris-buffered saline with Tween 20 (TBST) and incubated with Rabbit IgG (HRP) (GeneTex, CA, USA) secondary antibody for 1 h at room temperature. Membranes were washed and then detected by enhanced chemiluminescence substrate using Luminescence Imaging System (LAS-4000, GE, USA).
4
Western Blot Analysis of MMP-3 in hUCMSCs
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To prepare the cell lysates for Western blot, hUCMSCs were washed with PBS and lysed using M-PER mammalian protein extraction reagent (Thermo, IL, USA) with Halt protease inhibitor cocktail (Thermo, IL, USA), then placed in a centrifuge at 14,000 g for 10 min at 4°C to collect the precleared cell extracts. Protein concentration of the samples were determined using the Coomassie Plus (Bradford) protein assay reagent (Thermo, IL, USA) and Multimode microplate readers (Infinite 200, TECAN). Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck, Darmstadt, Germany). Membrane blocking solution 10% Fish gelatin blocking buffer (AMRESCO, OH, USA) was used to block PVDF membranes for 1 hour. Then membranes were incubated with the anti-human MMP-3 primary antibody (R&D systems, USA) at 1:1000 dilution at 4°C overnight, followed by washing with tris-buffered saline with tween 20 (TBST). The membranes were then incubated with goat anti-mouse secondary antibody at room temperature for an hour. The western blot data were detected by enhanced chemiluminescence substrate using Luminescence Imaging System (LAS-4000, GE, USA).
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