Brain homogenates were run on a 4–20% Tris–Glycine gel (Mini-protean TGX gel; Bio-Rad laboratories; #4561094) and transferred onto a nitrocellulose membrane (TransBlot® Turbo RTA mini nitrocellulose transfer kit; Bio-Rad laboratories; #1704270). The membranes were blocked in intercept blocking buffer (Li-Cor; #927-70001) for 1 h at room temperature, probed with primary antibodies against claudin-5 (1:250; Cell Signaling technology; #49564), occludin (1:500; BD Biosciences; #611091), VE-cadherin (1:1000; Abcam; ab205336), GFAP (1:1000; Sigma-Millipore; MAB360), and beta-actin (1:1000; Li-Cor; #926-42212 and #926-42210) diluted in intercept antibody diluent buffer (Li-Cor; #927-75001). Membranes were washed with PBS-T (0.1% Tween-20) and probed with Li-Cor IRDye secondary antibodies diluted in intercept antibody diluent buffer for 1 h at room temperature. The washed membranes were then imaged on a Li-Cor Odyssey CLx imaging system and blots were analyzed using the Image Studio Lite 5.2.5 software. Relative protein abundance was obtained by densitometry analysis. The signal intensity of all protein bands was normalized to the beta-actin loading control band (relative abundance).
Trans blot turbo rta mini nitrocellulose transfer kit
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo RTA Mini Nitrocellulose Transfer Kit is a tool designed for efficient and rapid protein transfer from polyacrylamide gels to nitrocellulose membranes. It provides a standardized and consistent method for Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Anti toxin a chicken igy, by Abnova (2 mentions)
Anti toxin b chicken igy, by Abnova (2 mentions)
Nu page lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (2 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Anti rac1, by Abcam (1 mentions)
Anti cdc42, by BD (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Autoradiography film, by Thomas Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Irdye 680, by LI COR (1 mentions)
Irdye 800, by LI COR (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
9 protocols using trans blot turbo rta mini nitrocellulose transfer kit
1
Western Blot Analysis of Tight Junction Proteins
2
Quantifying Protein Knockdown by Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used were polyclonal rabbit anti-Septin 7 (IBL, #18991), and monoclonal mouse anti-β-actin (Sigma-Aldrich, #A5316), anti-CDC42 (BD Transduction Laboratories, #610929), anti-RAC1 (Abcam, Cat# ab33186) as well as anti-ArpC3 p21-Arc (Clone26, #612234, BD Transduction Laboratories). Secondary antibodies were anti-mouse (Bio-Rad, #170-6516) and anti-rabbit (Bio-Rad, #170-6515) horseradish peroxidase-conjugated. To quantify knockdown efficiency by immunoblotting, siRNA treated HeLa cells were lysed with RIPA buffer containing protease inhibitor (Roche, #11836170001) for 30 min at 4°C. Protein quantification of lysates was done with the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, #23235) and 10 μg of total protein was loaded into NuPAGE 4%–12% Bis-Tris Protein Gels (Thermo Fisher Scientific, #NP0321BOX) or 12% SDS-PAGE gels. Proteins were transferred to a nitrocellulose membrane using the Trans-Blot® Turbo RTA Mini Nitrocellulose Transfer Kit (Bio-Rad, #1704270) and Trans-Blot© Turbo Transfer System (Bio-Rad, #1704150) or tank blotting system in Towbin Buffer (Tris 25 mM, Glycine 190 mM, 20% Methanol pH 8.3) during 1h at 300 mA at 4°C. Antibody detection was carried out with the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fischer Scientific, #34577) with actin as loading control.
3
Antigen Expression Analysis in Bacteria and Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For antigen expression in monomicrobial culture, the transformed YS1646 strains were grown overnight in LB with 50 μg/ml of ampicillin at 37°C in 0% CO2, centrifuged at 21,130 × g for 10 min, resuspended in PBS, and then mixed in with NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) according to the manufacturer’s instructions. For antigen expression in RAW 264.7 macrophages, infection was allowed to proceed for either 1 h or 24 h. Samples were then collected, centrifuged, resuspended in PBS, and mixed with sample buffer as described above. All samples were heated for 10 min at 70°C and then cooled on ice. Proteins were separated on a 4 to 12% Bis-Tris protein gel (Invitrogen) and transferred to nitrocellulose membranes using a Trans-Blot Turbo RTA mini-nitrocellulose transfer kit (Bio-Rad, Hercules, CA). For detection of TcdA5458–8130 and TcdB5461–7080, the membranes were incubated first with anti-toxin A chicken IgY (1:5,000; Abnova, Taipei, Taiwan) and anti-toxin B chicken IgY (1:10,000; Abnova) antibodies, respectively, followed by goat anti-chicken IgY conjugated to horseradish peroxidase (1:10,000; Thermo Fisher Scientific). Immunoreactive bands were visualized using the SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific) and autoradiography film (Denville Scientific, Holliston, MA).
4
TDP-43 Autoregulation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the TDP-43 autoregulation experiment, the cell lines were induced with 150 ng/ml doxycycline (Dox) for 48 h. In parallel a set of uninduced samples was processed. Cells were harvested, washed once with PBS and lysed with 300 μL of RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 25x Protease Inhibitor Cocktail (Roche) and Benzonase Nuclease (Novagen) for 5 min at 4°C. Supernatants were cleared of debris by 10 min centrifugation at 13000 rpm at 4 °C and total protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad). 2 μg of total cell lysates were supplemented with 4x Nupage loading buffer (+DTT, fc. 10mM) and separated over 4%–12% gradient SDS-PAGE gels, transferred to a 0.2 μm nitrocellulose membrane using the Trans-Blot Turbo RTA Mini Nitrocellulose Transfer Kit (Bio-Rad) and blotted with rabbit polyclonal anti-TDP-43 (10782-2-AP Proteintech), and mouse monoclonal anti-α-Tubulin (T5168 Sigma-Aldrich). After secondary antibody incubations, signals were detected by LI-COR secondary Antibodies (IRdye680 1:15000, IRdye800 1:15000) and visualized by Odyssey scanning (LI-COR) and quantified using Image Studio Lite (LI-COR).
5
Protein Extraction and Western Blot
6
Western Blot Protocol for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated in RIPA buffer supplemented with PMSF, protease inhibitor mixture, and sodium orthovanadate (RIPA Lysis Buffer System, sc24948, Santa Cruz, Dallas, TX). Samples were heated at 95°C for 5 min in SDS-PAGE sample buffer, loaded on a precast 4–20% polyacrylamide gradient gel (4561094, Biorad) and subjected to electrophoresis. Proteins were transferred onto a nitrocellulose membrane (Trans-Blot Turbo RTA Mini Nitrocellulose Transfer Kit, 1704270, BioRad) for 30 min using Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked with 5% milk in Tris-buffered saline with Tween 0.05% for 1 hr at room temperature. The membranes were exposed to primary antibody overnight at 4°C, rinsed and incubated with appropriate horseradish peroxidase-labelled secondary antibody for 1 hr at room temperature. The detection was performed by using SuperSignal West Femto (34096, ThermoFisher Scientific) and visualized with a G-Box Chemi-XT4 (SynGene, Cambridge, UK). GAPDH was used as reference protein.
7
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared or immunoprecipitation (IP) samples were eluted using Loading buffer, subsequently followed by heating at 95 °C for 5 min. SDS-PAGE was then implemented to separate proteins by molecular weight using the Mini-PROTEAN 3 electrophoresis module apparatus (Bio-rad). The gels were then subsequently transferred using the Pierce Power Blotter (Thermo Scientific), with reagents and instructions used from the Trans-Blot® Turbo™ RTA Mini Nitrocellulose Transfer Kit (Bio-rad), for 11 min, at a constant of 25 V. Membranes were then incubated with the appropriate Primary antibody overnight at 4 °C and then a minimum of 3 h at 4 °C with the appropriate Secondary antibody. Protein expression was analysed and detected using the Western Lightning® Plus-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer) and G:BOX Chemi XX6 gel doc system (Syngene). A list of antibodies is provided as a Supplementary Table.
8
In Vitro Antigen Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For antigen expression in vitro, the transformed YS1646 strains were grown overnight in LB at 37°C in 0% CO2. Overnight cultures were centrifuged at 21,130 × g for 10 min and resuspended in PBS. Six hundred seventy nanograms of each sample and 7 ng and 50 ng of positive controls were then mixed in with NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) according to the manufacturer’s instructions. All samples were heated for 10 min at 100°C and then cooled on ice. Proteins were separated on a 4% to 20% Bis-Tris protein gel (Invitrogen) and transferred to nitrocellulose membranes using a Trans-Blot Turbo RTA mini nitrocellulose transfer kit (Bio-Rad, Hercules, CA). For detection of TcdA5458–8130 and TcdB5461–7080, the membranes were incubated first with anti-toxin A chicken IgY (1:5,000; Abnova, Taipei, Taiwan) and anti-toxin B chicken IgY (1:10,000; Abnova) antibodies, respectively, followed by goat anti-chicken-IgY IgG conjugated to horseradish peroxidase (HRP; 1:10,000; Thermo Fisher Scientific). Immunoreactive bands were visualized using the SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific) and autoradiography film (Denville Scientific, Holliston, MA).
9
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology, Inc., Dallas, TX) according to the protocol supplied. Whole cell lysates (30 μg) were separated by SDS-PAGE, transferred to nitrocellulose through Trans-Blot Turbo RTA Mini Nitrocellulose Transfer Kit (Bio-Rad, Hercules, CA, USA). Membranes were subjected to immunoblot analyses using primary antibodies against phosphorylated RB (Ser780) #8180 1:1,000 (Cell Signaling Technology, Danvers, MA), phosphorylated RB (Ser807/811) #8516 1:1,000 (Cell Signaling Technology), RB (IF-8) #sc102 1:1,000 (Santa Cruz Biotechnology, Inc.), Erα (F-10) #sc-8002 1:200 (Santa Cruz Biotechnology, Inc.), PLK1 (208G4) #4513 (Cell Signaling Technology) 1:1,000, β-actin (13E5) rabbit mAb #4970 (Cell Signaling Technology) 1:5,000, PARP (46d11) rabbit mAb #9532 (Cell Signaling Technology), GAPDH #sc-32233 1:10,000 (Santa Cruz Biotechnology, Inc.), HRP-conjugated anti-rabbit and anti-mouse were used as secondary antibodies (Bio-Rad). Immunoreactive proteins were visualized by enhanced chemiluminescence using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). Membranes were cut horizontally to probe with multiple antibodies. Films were imaged using Brother MFCL2710DW (Brother) at 300 dpi.
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!