The PVDF membrane was blocked in 5% w/v nonfat dry milk in 1X TBS and 0.1% Tween 20 (TBST, Bio-Rad) for 1 h prior to antibody incubations. Actin was probed using a pan-actin D18C11 Rabbit mAb (1:1000, Cell Signaling Technology) diluted in 5% w/v BSA and 1X TBST (Bio-Rad), followed by an anti-rabbit IgG HRP-linked Ab (1:2000, Cell Signaling Technology) diluted in 5% w/v nonfat dry milk and 1x TBST (Bio-Rad), both for 1 h at room temperature. MyoD was detected using the same antibody as used for immunofluorescence. The biotinylated ladder was probed using an anti-biotin HRP-linked Ab (1:2000, Cell Signaling Technology) diluted in 5% w/v nonfat dry milk and 1X TBST (Bio-Rad) for 1 hour at 25C. The membrane was washed three times with TBST for 15 min between each antibody incubation.
Tbs t
Manufactured by Bio-Rad
Sourced in United States
TBS-T is a buffer solution used in various biochemical and molecular biology techniques. It is a mixture of Tris-buffered saline (TBS) and Tween-20, a non-ionic detergent. TBS-T is commonly used as a washing buffer in procedures such as Western blotting, ELISA, and immunohistochemistry to remove unbound or non-specific proteins or antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Tbs t, by Cell Signaling Technology (6 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Chemidoc imaging system, by Bio-Rad (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Wet electroblotting system, by Bio-Rad (4 mentions)
Sds page gel, by Bio-Rad (4 mentions)
Anti rabbit igg conjugated with horseradish peroxidase, by Cell Signaling Technology (4 mentions)
Tris glycine buffer, by Bio-Rad (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Pierce ecl western blotting substrate, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
β actin hrp, by Santa Cruz Biotechnology (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Protease and phosphatase, by Thermo Fisher Scientific (2 mentions)
Ponceau s solution, by Merck Group (2 mentions)
42 protocols using tbs t
1
Western Blot Protein Detection Assay
2
Mammary Tissue Lysis and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole mammary tissue sample (50 mg) was weighed from the most distal portion of the #4 inguinal mammary gland and lysed in RIPA lysis buffer (Thermo-Fisher Scientific) with protease and phosphatase (Thermo-Fisher Scientific) inhibitors at 100x dilution in Precellys 2mL tissue homogenizing mixed beads kit (Cayman Chemical) using a Precellys homogenizer at 6500 rpm (2 cycles of 15 sec motion-15 sec rest). Immunoblots were carried out with 40µg of total protein. Membranes were blocked in TBS-T (Bio-Rad, with 0.05% Tween20) with 4% milk (Bio-Rad, #170-6404) and incubated with primary antibodies in TBS-T at 4°C overnight. Membranes were stained with Ponceau-S solution (Sigma-Aldrich) to confirm equal loading. Anti-CCN6 (Santa Cruz Biotechnology, #SC-25443) was the primary antibody.
3
Mammary Tissue Lysis and Immunoblotting
4
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were detached by manual scraping, collected by centrifugation and lysed in NP-40 cell lysis buffer (150 mM NaCl, 50 mM Tris, 1% NP-40) supplemented with protease and phosphatase inhibitors. Lysates were centrifuged at max speed for 15 min to remove cell debris. Protein concentrations were determined by Bradford assay (Bio-Rad, Richmond, CA, USA) using bovine serum albumin (BSA; 2 mg/mL) as a standard. The quantified protein samples were stored at −20 °C for further use. 20 µg protein lysate was run on 12% polyacrylamide gel. Blotting was performed by using 0.45 µm Nitrocellulose membrane (Bio-Rad) and wet transfer system (GE TE 22 Mini Tank Transfer Unit) overnight at 4 °C and 16 V. Membranes were blocked with 5% non-fat dry milk in TBS-T (Bio-Rad). Primary antibody binding was performed overnight at 4 °C with gentle agitation. Membranes were washed with TBS-T and incubated with secondary antibody at RT for 2 h. After the final wash, membrane was treated with Clarity™ ECL Western Blotting Substrate (Bio-Rad) and visualized via ChemiDoc Imaging System (Bio-Rad, Richmond, CA, USA).
5
Western Blotting of Uterine and Oviduct Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates (40 µg per lane) from uteri or oviducts were loaded on a 4–15% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA), separated in 1X Tris-Glycine Buffer (Bio-Rad), and transferred to PVDF membranes via a wet electro-blotting system (Bio-Rad), all according to the manufacturer’s directions [20 (link)]. PVDF membranes were blocked for 1 hour in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T, Bio-Rad), then incubated overnight at 4 °C with antibodies listed in Supplementary Table 2 in 5% BSA in TBS-T. Blots were then probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1:5000, Cell Signaling Technology, Danvers, MA, USA) in 5% BSA in TBS-T for 1 hr at room temperature. Signal was detected with the Pierce™ ECL Western Blotting Substrate (Millipore, Billerica, MA, USA), and blot images were collected with a Bio-Rad ChemiDoc imaging system.
6
Western Blot Analysis of MET, CPD, and GAPDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and harvested in RIPA lysis buffer (Thermo Fisher Scientific, 89900) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 87786). Twenty micrograms of cell lysate was denatured in Laemmli SDS-Sample Buffer (Boston BioProducts, BP-110R) and loaded on a precast 4–15% Mini-Protean TGX Stain-Free gel (Bio-Rad). After electrophoresis and transfer, the PVDF membrane (Bio-Rad) was blocked in 5% nonfat milk with TBST (Bio-Rad) at room temperature for 1 h and incubated with primary antibody overnight. MET (3127, 1:1000) antibody was purchased from cell signaling technology. CPD (A305-514A-M, 1:1000) and GAPDH (MA5-15738, 1:5000) antibodies were purchased from Thermo Fisher Scientific. The membranes were then washed in TBST three times, followed by incubation with goat anti-rabbit/Mouse secondary antibodies (Cell Signaling Technology, 1:2000–1:5000) for 1 h at room temperature. After washing with TBST, the membranes were developed by films. All western blots were repeated twice. Image quantification was performed in ImageJ.
7
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates (40 μg per lane) were loaded on a 4–15% SDS–PAGE gel (Bio-Rad), separated in 1X Tris-Glycine Buffer (Bio-Rad), and transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore) via a wet electro-blotting system (Bio-Rad), all according to the manufacturer’s directions and as described previously (109 (link)). PVDF membranes were blocked for 1 h in 5% non-fat milk (Bio-Rad) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T; Bio-Rad), then incubated overnight at 4°C with antibodies listed in Table S2 in 5% BSA in TBS-T. Blots were then probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1:5,000; Cell Signaling Technology) in 5% BSA in TBS-T for 1 h at room temperature. Signal was detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore), and blot images were collected with a Bio-Rad ChemiDoc imaging system.
8
Quantification of CD3ζ Protein Levels
9
Western Blotting for Protein Profiling
10
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using a lysis buffer (Invitrogen) with protease and phosphatase inhibitors (Roche), and protein concentrations in the cell lysates were determined using Quick Star Bradford dye reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were subjected to SDS-PAGE gel electrophoresis, and then the protein-containing gel was transferred to a PVDF membrane (Millipore). The membranes were blocked with TBST (Bio-Rad) containing 5% (w/v) skim milk for 30 min and then incubated with primary antibodies overnight at 4 °C. On the following day, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein expression signals were enhanced using a chemiluminescence detection solution (Bio-Rad) and captured using the Fusion Solo imaging system (Vilber Lourmat, Marne-la-Vallee, France). The full-length original gels and blots were included in the supplemental experimental procedure, and the antibodies used are listed in Table S2 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!