Immunoblotting protein samples were prepared using 4x XT sample buffer and 20x XT reducing reagent (Biorad). Samples were boiled for 10 minutes at 95°C before being spun down and added to protein gels. To separate proteins by SDS-PAGE, Criterion pre-cast 4-12% gradient Bis-tris gels were used and run for 2 hours at 120V. Proteins were transferred to PVDF membranes by semi-dry transfer at 25V for 45 minutes. Protein laden PVDF membranes were blocked for 1 hour in 5% milk dissolved in TBST. Membranes were briefly rinsed with TBST and incubated with primary antibodies diluted in 5% BSA TBST for either 1 hour at room temperature or overnight at 4°C depending on the antibody. Following primary incubation blots were rinsed with TBST 3 times for 10 minutes each and placed in HRP-conjugated Igg secondary diluted in 5% milk TBST for 1 hour at room temperature. Blots were rinsed 3 times for 10 minutes each and imaged using SuperSignal HRP chemiluminescent substrate (ThermoFisher Scientific).
Xt sample buffer
Manufactured by Bio-Rad
Sourced in United States
The XT sample buffer is a laboratory reagent used to prepare protein samples for analysis. It is designed to maintain the native structure and properties of proteins during sample preparation and handling. The buffer helps to solubilize and denature proteins, making them suitable for subsequent analytical techniques such as gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Xt reducing agent, by Bio-Rad (10 mentions)
Xt mops buffer, by Bio-Rad (4 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Irdye 800cw goat anti rabbit, by LI COR (3 mentions)
Reducing agent, by Bio-Rad (3 mentions)
Xt mes, by Bio-Rad (2 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Benzonaze, by Merck Group (2 mentions)
Pierce ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Mouse anti β actin antibody, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Dc protein assay reagent, by Bio-Rad (2 mentions)
43 protocols using xt sample buffer
1
Immunoblotting for Protein Detection
2
APP-derived Fragments Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For APP-derived fragments (C99 and APP) analysis, the parieto-temporal cortex extracts used for determining Aβ levels were mixed with 4x XT sample buffer (Bio-Rad), boiled for 5 min at 95 ºC, resolved onto a Criterion TM XT precast gel 4-12% Bis-Tris (Bio-Rad) using XT MES (Bio-Rad) running buffer and transferred to nitrocellulose membranes.
In the rest of cases, protein samples were mixed with 6x Laemmli sample buffer, boiled for 5 min at 95 ºC, resolved onto SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
In all cases, membranes were then blocked with 5% milk in TBS and incubated overnight with the following primary antibodies: mouse monoclonal 6E10 (reactive to amino acids 1-17 of Aβ peptide, 1:1000, BioLegend), mouse monoclonal anti-p-Tau One TM software v.4.6.3 (Bio-Rad) was used for protein quantification.
In the rest of cases, protein samples were mixed with 6x Laemmli sample buffer, boiled for 5 min at 95 ºC, resolved onto SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
In all cases, membranes were then blocked with 5% milk in TBS and incubated overnight with the following primary antibodies: mouse monoclonal 6E10 (reactive to amino acids 1-17 of Aβ peptide, 1:1000, BioLegend), mouse monoclonal anti-p-Tau One TM software v.4.6.3 (Bio-Rad) was used for protein quantification.
3
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stimulated cells were washed twice with ice-cold PBS and lysed in Tris-buffered saline containing 1 % Nonidet P-40, 60 mM octyl-β-glucoside, 2 mM phenylmethylsulfonylfluoride, 10 μg/ml aprotinin, 2 μg/ml leupeptin and pepstatin A, 50 mM NaF, and 1 mM sodium orthovanadate for 30 min on ice. The lysates or immunoprecipitates were centrifuged for 15 min at 14000 g. The samples for polyacrylamide gel electrophoresis (PAGE) analysis were mixed with 4× XT sample buffer (Bio-Rad, Hercules, CA) and boiled for 4 min and separated on 10 % sodium dodecylsulfate-PAGE and transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated with anti-human ZO-1 mAb (Zymed), anti-human OCLN rabbit mAb (Zymed), anti-human E-cad rabbit mAb (Cell Signaling), and anti-human PCDH1 mAb (Santa Cruz) as a primary antibody and an appropriate secondary horseradish peroxidase-conjugated antibody (Fig. 6 ). Signals were detected using enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).
4
Western Blot Analysis of NCAM2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To 12.5 μL (50 μg) of
each sample, 4.2 μL of 4× XT sample buffer (Bio-Rad) supplemented
with 10% 2-mercaptoethanol was added, followed by 5 min of heating
at 95 °C. 15 μL (45 μg) of each sample was loaded
on 4–12% Criterion XT Bis-Tris Protein Gels (Bio-Rad) and separated
by SDS-PAGE, after which they were transferred onto 0.2 μM PVDF
membranes (Bio-Rad) to be analyzed by western blotting. Primary antibodies
used were anti-NCAM2 (1:2,000, Santa Cruz Biotechnology) and anti-Actin
(1:5,000, Sigma-Aldrich). Secondary antibodies used were anti-Mouse
IgG, HRP-linked (1:10,000, Cell Signaling) and anti-Rabbit IgG, HRP-linked
(1:10,000, Cell Signaling). Proteins were detected with Pierce ECL
Plus Western Blotting Substrate (Thermo Fisher Scientific) and an
AI600 Imager (GE Healthcare). Images were further processed and visualized
with ImageJ version 1.52 and Adobe Illustrator 2023. For proteome
analysis, 30 μL (15 μg) of SH-SY5Y whole cell lysate of
each sample was analyzed by SDS/PAGE followed by western blotting
as described above with minor changes. Primary antibodies used were
anti-NCAM2 (1:2000) and anti-Actin (1:5000). Secondary antibodies
used were polyclonal Goat Anti-mouse Immunoglobulin/HRP (1:2000, Agilent)
and anti-Rabbit IgG, HRP-linked (1:10,000).
each sample, 4.2 μL of 4× XT sample buffer (Bio-Rad) supplemented
with 10% 2-mercaptoethanol was added, followed by 5 min of heating
at 95 °C. 15 μL (45 μg) of each sample was loaded
on 4–12% Criterion XT Bis-Tris Protein Gels (Bio-Rad) and separated
by SDS-PAGE, after which they were transferred onto 0.2 μM PVDF
membranes (Bio-Rad) to be analyzed by western blotting. Primary antibodies
used were anti-NCAM2 (1:2,000, Santa Cruz Biotechnology) and anti-Actin
(1:5,000, Sigma-Aldrich). Secondary antibodies used were anti-Mouse
IgG, HRP-linked (1:10,000, Cell Signaling) and anti-Rabbit IgG, HRP-linked
(1:10,000, Cell Signaling). Proteins were detected with Pierce ECL
Plus Western Blotting Substrate (Thermo Fisher Scientific) and an
AI600 Imager (GE Healthcare). Images were further processed and visualized
with ImageJ version 1.52 and Adobe Illustrator 2023. For proteome
analysis, 30 μL (15 μg) of SH-SY5Y whole cell lysate of
each sample was analyzed by SDS/PAGE followed by western blotting
as described above with minor changes. Primary antibodies used were
anti-NCAM2 (1:2000) and anti-Actin (1:5000). Secondary antibodies
used were polyclonal Goat Anti-mouse Immunoglobulin/HRP (1:2000, Agilent)
and anti-Rabbit IgG, HRP-linked (1:10,000).
5
Co-immunoprecipitation of Amyloid-beta
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly prepared subcellular fractions (or total microsome preparations) were incubated with a cross-linker solution of dithiobis(succinimidyl propionate) (DSP, Pierce), to a final concentration of 2 mM for 30 min at RT, after that Stop Solution (1 M Tris pH 7.5) was added to a final concentration of 20 mM for 15 min. Cross-linked subcellular fractions (or total microsomes) samples were incubated with anti-V5 agarose affinity gel (Sigma) for 16 h at 4 °C for the immunoprecipitation procedure. After ice-cold PBS washes (6 times 5,000 g, 5 min each), the immunoprecipitated proteins were eluted with 4 × Tricine sample buffer (Bio-Rad) containing β-mercaptoethanol by 10 min boiling, electrophoresed on Tris-Tricine gel 16.5% (precasted gels, Bio-Rad). The semidry transfer blot was performed in nitrocellulose membrane filter 0.22 μm and the filter boiled in PBS and subjected to WB analyses (mAb 6E10 anti-Aβ/APP and mAb anti-V5-HRP). Co-IP of small AβOs and intrabody was analysed in Tris-Tricine gels 16.5% for a better resolution of low MWs (<50 kDa) WB bands. The full-length APP (~\n110 kDa) was analysed in Tris-glycine HCl gels 10% or in Criterion Bis-Tris XT Gels 4–12% (Bio-Rad) upon elution and boiling of immunoprecipitated proteins with the appropriate sample buffers (respectively 4 × Laemmli Sample buffer or 4 × XT sample buffer Bio-Rad with β-mercaptoethanol).
6
Protein Profiling of Milk Fat Globule Membrane
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein profile was investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were normalized according to protein content (4 mg/mL) and then dissolved in sample buffer (4× XT Sample Buffer, Bio-Rad, Hercules, CA, USA) containing 5mL/100mL of Tris (2-carboxyethyl) phosphine hydrochloride (TCEP.HCl). The mix, with a final protein concentration of 1mg/mL, was heated at 95 °C for 3 min and then loaded on a 12% Bis-Trispolyacrilamide gel (Criterion_XT, Bio-Rad). Moreover, the protein pattern of MFGM isolated from selected BM and BS was investigated by means of 3–8% Tris-Acetate gels (Criterion_XT, Bio-Rad), which allowed for better separation and discrimination of high-molecular-weight proteins (50–250 kDa). Electrophoresis was conducted for 1 h at a constant voltage of 150 V. Finally, the 2-D gels were stained with a dye (Bio-Safe™ Coomassie G-250, Bio-Rad) and scanned with a Versa-Doc image system (Bio-Rad).
7
Assay of IRF3 Dimerization under Semi-Native Conditions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IRF3 dimerization was assayed under semi-native conditions. Cells were lysed in ice-cold Pierce RIPA lysis buffer (Thermo Scientific) supplemented with 10 mM NaF, 1x complete protease cocktail inhibitor (Roche) and 5 IU mL−1 benzonaze (Sigma). Protein concentration was determined using a BCA protein assay kit (Thermo Scientific). Whole-cell lysates were mixed with 1x XT Sample Buffer (BioRad); samples were neither reduced nor heated before separation was done on 4–20% Criterion TGX precast gradient gels (BioRad) by SDS-PAGE electrophoresis. Each gel was run initially for 15 min at 70 V and 15 min at 120 V. Transfer onto PVDF membranes (BioRad) was done using a Trans-Blot Turbo Transfer system for 7 min. Membranes were blocked for 1 h with 5% skim-milk (Sigma Aldrich) at room temperature in PBS supplemented with 0.05% Tween-20 (PBST). Membranes were probed overnight at 4 °C with the following specific primary antibody in PBST: anti-IRF3. After three washes in PBST, secondary antibodies, peroxidase-conjugated F(ab)2 donkey anti-rabbit IgG (H + L) (1:10,000) (Jackson Immuno Research) were added to the membrane in PBST 1% milk for 1 h at room temperature. All membranes were washed three times and exposed using either the SuperSignal West Pico PLUS chemiluminescent substrate or the SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific).
8
Recombinant Baculovirus Expression in Insect Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Insect cell culture, recombinant bacmid generation, and recombinant baculovirus generation and amplification are detailed in the Supplementary Methods. During the screening test, a multiplicity of infection (MOI) of 0, 3, 5, 10 was used to transfect 4 mL of Sf9 insect cell culture. MOI is the ratio of the number of virus particles to the number of cells in cell culture. A 4 mL volume of culture was collected after post-transfection at 48, 72, and 96 h. A 100 μL volume of cell culture was pelleted and lysed with 50 μL of SDS-PAGE Loading Buffer (1X XT Sample Buffer [Bio-Rad #161-0791], 715 mM β-mercaptoethanol). All samples were heated up at 96 °C for 10 min before loading on SDS-PAGE. Samples were analyzed by SDS-PAGE on 4–12% acrylamide Bis–Tris gels (Invitrogen # NP0329BOX) in 1X MOPS-SDS running buffer (Invitrogen, # NP0001). For western blotting, the primary antibody, anti-pGC-A, was purchased from R&D Systems (# MAB4860); the secondary antibody, HRP-conjugated anti-mouse IgG, was procured from Jackson ImmunoResearch (# 515–035-062). Original blots/gels are in Supplementary Fig. S7 .
9
Analyzing SARS-CoV-2 S Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze S protein expression in HEK293T cells, the plasmids encoding V5-tagged S protein were transfected into these cells, using Lipofectamine 2000. Four hours later, the medium was replaced by medium with 2% FCS and the cells were incubated for another 48 h. Cells were washed once with PBS and lysed in RIPA buffer supplemented with protease inhibitor cocktail (both from Thermo Fisher Scientific).
For analysis of S protein incorporation into pseudoparticles, a volume of 600 μl of S-pseudotyped MLV virus was loaded onto a 20% (w/v) sucrose cushion (volume 50 μl) and subjected to high-speed centrifugation (25,000 g for 120 min at 4°C). Thereafter, 630 μl of supernatant was removed and the residual volume was mixed with 30 μl loading dye mastermix, consisting of RIPA buffer supplemented with protease inhibitor cocktail (both from Thermo Fisher Scientific) and 1x XT sample buffer containing 1x XT reducing agent (both from Bio-Rad). The samples were heated for 5 min at 95°C and subjected to SDS-PAGE and immunoblotting, as above. Antibody details can be found inS4 Table .
For analysis of S protein incorporation into pseudoparticles, a volume of 600 μl of S-pseudotyped MLV virus was loaded onto a 20% (w/v) sucrose cushion (volume 50 μl) and subjected to high-speed centrifugation (25,000 g for 120 min at 4°C). Thereafter, 630 μl of supernatant was removed and the residual volume was mixed with 30 μl loading dye mastermix, consisting of RIPA buffer supplemented with protease inhibitor cocktail (both from Thermo Fisher Scientific) and 1x XT sample buffer containing 1x XT reducing agent (both from Bio-Rad). The samples were heated for 5 min at 95°C and subjected to SDS-PAGE and immunoblotting, as above. Antibody details can be found in
10
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
786-O cells (5 × 105) were seeded into 6-well plate and treated as desired. Afterwards, each well was pelleted and lysed in 80 μL of ice-cold Pierce RIPA lysis buffer (Thermo Scientific) supplemented with 10 mM NaF, 1× protease inhibitors (Roche), and 5 IU/mL benzonaze (Sigma), respectively. The protein concentration was determined and equilibrated using a Pierce BCA protein assay kit (Thermo Scientific). 25 µg of whole-cell lysates was denatured for 5 min at 95 °C in the presence of 1× XT sample buffer and 1× XT reducing agent (both Bio-Rad). Separation of samples was performed by SDS-PAGE on 4−20% Criterion TGX precast midi protein gels (Bio-Rad) at 75 V until the dye front reached the separating gel and then at 120 V until it reached the bottom of the gel. Precision Plus Dual Color protein ladder (Bio-Rad) was used as separation control. Proteins exceeding a molecular weight of 200 kDa (such as TET2) were separated using 4–8% Criterion XT Tris-acetate precast midi protein gels in the absence of SDS, employing a Tris-acetate buffer system at pH 7.0 (Bio-Rad).
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!