M.smegmatis strains were grown in Hartmans-de Bont liquid media to an OD of 0.8 and induced sequentially with theophylline (2mM) and tetracycline (5ng/ml). Acetamide-induced ParAB (a gift from Dagmara Jakimowicz) was included as a control. Bacterial cells following induction were harvested by centrifugation at 3000rpm for 10 minutes. The cell pellet was washed and re-suspended in PBS and complete protease inhibitors (Roche diagnostics), followed by rupture in a ribolyser using 0.1mm silica beads (MP Biomedicals). The lysate was recovered after centrifugation at 13,000rpm for 30 minutes. Total protein concentration was determined using the Pierce BCA assay kit (Thermo Fisher Scientific) and 20μg whole cell lysates were boiled and separated on a 12% polyacrylamide gel (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane (GE healthcare). The membrane was blocked in TBST (0.05% Tween 20) containing 3% BSA (Roche diagnostics). The membrane was probed with 1:2000 rabbit polyclonal anti-par A (absorbed against M. smegmatis ΔparAB) and affinity-purified anti-par B antibodies (gifts from Dagmara Jakimowicz) overnight at 4°C. The membrane was subsequently incubated with 1:5000 goat anti-rabbit IgG antibody (Thermo Fisher Scientific) for 1 hour at room temperature and developed using the SuperSignal West Femto kit (Thermo Fisher Scientific).
Goat anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-rabbit IgG antibody is a secondary antibody used in various immunoassays and detection techniques. It binds to the Fc region of rabbit primary antibodies, allowing for the detection and visualization of target proteins or analytes.
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Lab products found in correlation
Goat anti mouse igg antibody, by Thermo Fisher Scientific (3 mentions)
Supersignal west femto kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Plan apochromat 63 1.4 oil dic lens, by Zeiss (2 mentions)
Dylight 550 goat anti mouse igg antibody, by MyBioSource (2 mentions)
Rabbit monoclonal anti mcm5 or anti mcm3 antibody, by Abcam (2 mentions)
Cell observer spinning disk fluorescent microscope, by Zeiss (2 mentions)
Fluorescence microscope, by Leica (2 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
0.1 mm silica beads, by MP Biomedicals (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Ki 67 primary antibodies, by Abcam (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mouse gapdh antibody, by Abcam (1 mentions)
27 protocols using goat anti rabbit igg antibody
1
ParAB Protein Expression Regulation
2
Evaluating PAEC Proliferation via Ki-67
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Ki-67 immunofluorescence staining was performed using the labeled streptavidin–biotin method to evaluate the proliferation of PAECs. After being washed three times with phosphate buffer saline (PBS), the PAECs were sequentially fixed in 4% paraformaldehyde at 4°C for 20 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. The cells were then incubated with the Ki-67 primary antibodies (1:500 dilution, Abcam, Cambridge, MA, United States) at 4°C overnight, and then stained with goat anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific, Alexa Fluor 488) at 37°C for 1 h. Finally, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:500, Abcam). The Ki-67 immunostaining of PAECs were detected by a fluorescence microscope (Zeiss, Heidenheim, Germany). The percentage of Ki-67-positive cells was indicated as the proliferation rate of PAECs. Measurements were repeated three times.
3
MCMV Infection-Induced Inflammasome Activation
4
Protein Expression Analysis of 2D and 3D Cell Cultures
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On day 5, cells cultured in 2D and 3D systems were washed twice in PBS and lysed using the M-PER® Mammalian Protein Extraction Reagent (Thermo, 75801) supplemented with proteinase inhibitor cocktail (Roche, 04693116001) for 30 min on ice. Whole-cell lysates were harvested from the supernatant by centrifugation at 12,000 g for 15 min. Lysates were electrophoresed in SDS-polyacrylamide gels (Beyotime Technologies, P0012A) and transferred to PVDF membranes (Millipore, IPVH00010). Primary antibodies used in this study included: anti-CD133 (Millipore, W6B3C1), anti-MGMT (Cell Signaling, 2739S), anti-Sox2 (Cell Signaling, 3579P), anti-Nanog (Cell Signaling, 4903), anti-Oct4 (Cell Signaling, 2750), anti-GAPDH (Bioworld, AP0063) and anti-β-tubulin (Cell Signaling, 2146S). A secondary HRP-linked goat anti-rabbit IgG antibody (Thermo, 31210) or goat anti-mouse IgG antibody (Thermo, 31431) was used as appropriate. Results were visualized using SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific).
5
Quantitative Protein Analysis via SDS-PAGE
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To extract total proteins, the transfected cells were lysed with RIPA lysis buffer (Cat#: C500005, Sangon) containing 5 mM EDTA and PMSF. The extracted proteins were subjected to quantitative analysis using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Ten micrograms of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk for 2 h at room temperature. Next, the membrane was incubated with diluted primary antibodies against SORBS1 (1:1000, Cat#: PA5-55656, rabbit human; Thermo Fisher Scientific Inc.), Bax (Cat#:ab32503, 1:2000, Abcam), Bcl-2 (Cat#:ab32124, 1:1000, Abcam), GAPDH (1:1000, Cat#: A300-639A, rabbit human; Thermo Fisher Scientific Inc.), p-JNK (Cat#: ab124956, 1:10,000, Abcam), and JNK (Cat#: ab208035, 1:10,000, Abcam) overnight at 4°C. Subsequently, the membrane was incubated with diluted goat anti-rabbit IgG antibody (1:5000, Cat#: A32731, Thermo Fisher Scientific Inc.) at room temperature for 2 h. Finally, the protein bands were visualized using a hypersensitive enhanced chemiluminescence kit (Cat#: C510043, Sangon) [28 (link)].
6
Occludin and Claudin-1 Protein Expression
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The protein expressions of occludin and claudin-1 were determined by Western blotting analysis. The total protein of the jejunum tissue was extracted using a total protein extraction kit (Beyotime Biotechnology, Shanghai, China). Twenty micrograms of protein from each group were separated by SDS-PAGE on 10% polyacrylamide gels. Separated proteins were then electrotransferred onto nitrocellulose membranes (Beyotime Biotechnology). Membranes were blocked with 5% nonfat dry milk in the TBS-Tween 20 buffer. Next, the membrane was incubated overnight at 4 °C with primary antibody. The primary antibodies were mouse monoclonal anti-claudin-1 (Huaan Biotechnology, Hangzhou, China; 1:500) and rabbit polyclonal anti-occludin (Huaan Biotechnology; 1:1000). Membranes were subsequently probed with rabbit monoclonal anti-beta actin (Abcam, Cambridge, UK; 1:1000). The secondary antibodies were goat anti-Mouse IgG Antibody (Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Massachusetts, USA). Membranes were incubated with an enhanced chemiluminescence detection kit (Beyotime Biotechnology). Images were taken (GS-700, Bio-Rad, Hercules, CA, USA) and were quantified by densitometry with Image J software.
7
Validating MLL1-ZC3H13 Fusion Expression
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The isolated clones (V5‐tagged) were tested for the expression of MLL1‐ZC3H13 fusion construct by flow cytometry using the anti‐V5 antibody. Along with clones, a parental control and vector control were also used for the validation assays. Briefly, for flow cytometry, the single‐cell population of clones and controls were fixed and permeabilized using reagents from the kit following the manufacturer's suggestion (BD Cytofix/Cytoperm, BD Biosciences, Mississauga, Canada; 554714). The cells were stained with anti‐V5 antibody (Abcam, Toronto, Canada, AB9116) and then with secondary goat anti‐rabbit IgG antibody conjugated with AF488 (Thermo Fisher Scientific, A11008). The data were subsequently analyzed by flowjo ® software (FlowJo LLC., Ashland, OR, USA, version 9.9 for Mac). The stemness of the clones was also assessed by flow cytometry following direct staining of cells without fixation or permeabilization using FITC‐conjugated mouse anti‐Human CD44 (BD Biosciences, 555478) along with Isotype control (FITC‐conjugated Mouse IgG2b Κ‐BD Biosciences, 555742).
8
Western Blot Analysis of E2 Protein
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Cells were washed with PBS and then lysed in Laemmli loading buffer (50 mM Tris-HCl, pH 6.8, 15% glycerol, 2% sodium dodecyl sulfate (SDS), 0.05% bromophenol blue, and 5 mM DTT), sheared through a 27-gauge needle (BD, Heidelberg, Germany) and heated for 10 min at 95 °C. The proteins were separated on a 10% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (GE Healthcare, IL, USA). After blocking with 10% Roti block (Roth, Karlsruhe, Germany), incubation with the primary antibody rabbit anti-E2 YF 7/28/87 (kindly provided by C. M. Rice, Rockefeller University, New York, NY, USA) at 1:10,000 in PBS overnight at 4 °C followed. The secondary HPO-coupled goat anti-rabbit IgG antibody (Pierce, Thermo Fisher, Waltham, MA, USA) was applied at 1:10,000 in PBS for 1 h at room temperature. For detection, the SuperSignal™ West Femto Kit (ThermoFisher Scientific) was used according to the manufacturer’s protocol.
9
Immunofluorescence Analysis of Cell Markers
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For immunofluorescence analysis cells were fixed with CellSave reagent, permeabilized with 0.1% Triton X-100 (Merck, Darmstadt, Germany), and stained for nucleic acid (DAPI; F. Hoffmann-La Roche, Basel, Switzerland), CK (clone C11, Alexa Fluor 488 conjugated, Cat#: GTX11212, GeneTex, Irvine, United States or clones C11/AE1/AE3, TRITC conjugated, Cat#: CKALLRMB000S, Aczon, Monte San Pietro, Italy), CD45 (clone 35-ZS, Alexa Fluor 647 conjugated, Cat#: sc-1178 AF647, Santa Cruz Biotechnology, Dallas, TX, United States), and caspase cleaved cytokeratin (M30 Cytodeath, FITC conjugated, Cat#: 10800, VLVbio, Nacka, Sweden), Ki67 (clone D3B5, Cat#: 9129S, Cell Signaling Technology, Danvers, MA, USA), phosphorylated Akt (clone D9E, Cat#: 4060, Cell Signaling Technology), or phosphorylated mTOR (clone D9C2, Cat#5536, Cell Signaling Technology). For Ki67 analysis a goat anti-rabbit IgG antibody (Cat# A-11012, Thermo Fisher Scientific, Waltham, MA, USA), for phosphorylated Akt and mTOR a donkey anti-rabbit IgG antibody (Cat# A-21206, Thermo Fisher Scientific) was used as the secondary antibody.
10
Immunoprecipitation of HSP47 Protein Complexes
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KEL FIB cells were lysed in RIPA buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and 10 mM N-ethylmaleimide (Sigma-Aldrich) and then spun at 16,000g for 15 minutes at 4 C. The lysate was incubated with anti-HSP47 antibody (1:50) (Santa Cruz Biotechnology) at 4 C on a shaker at 20 r.p.m. overnight. The coimmunoprecipitated protein complex was then isolated by adding protein G PLUS-Dynabeads (Santa Cruz Biotechnology). After an additional overnight rotation at 4 C with the beads, the coimmunoprecipitate was collected by spin magnet (Thermo Fisher Scientific) and washed twice with RIPA buffer. The pellet was eluted by boiling in 2Â sample buffer (Bio-Rad Laboratories) supplemented with b-mercaptoethanol (Sigma-Aldrich) and spun for 1 minute at 13,000 r.p.m. at 4 C. Samples were loaded on 7 % (w/v) SDS-PAGE, electrophoresed, and transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific). The primary antibodies used were as follows: anti-ACTB antibody (1:1000) and anti-ubiquitin antibody (1:1000) (Santa Cruz Biotechnology). The secondary antibodies used were goat anti-mouse IgG antibody (1:5000) and goat anti-rabbit IgG antibody (1:5000) (Thermo Fisher Scientific), and the signal was detected using an enhanced chemiluminescence kit (Bio-Rad Laboratories).
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