Transiently transfected cells were washed twice with PBS and lysed in NP-40 lysis buffer (P0013F; Beyotime) supplemented with protease inhibitor (P1005; Beyotime). Whole cell lysate was firstly precleared with protein A/G slurry (sc-2003; Santa Cruz) as follows: After centrifugation at 1,000 × g for 5 min at 4°C, supernatant was incubated with 1 μg of mouse anti-FLAG MAb, anti-HA MAb or anti-MYC MAb and 50 μl of protein A/G slurry (sc-2003; Santa Cruz) and incubated with rotation for 4 h at 4°C. Immunoprecipitated samples collected by centrifugation were washed with NP-40 lysis buffer four times. The final pellet was dissolved in 4 × SDS loading buffer (P1016; solarbio) for SDS-PAGE and Western blotting. Immunoprecipitations and the whole-cell lysates were probed with mouse anti-FLAG MAb, rabbit anti-FLAG PcAb and mouse anti-HA MAb or antibodies indicated in the figure legends. HRP-labeled goat anti-mouse light chain as secondary antibody for eliminating heavy chain interference.
Sds loading buffer
Manufactured by Solarbio
Sourced in China
2 × SDS Loading Buffer is a laboratory reagent used to prepare protein samples for SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). It is a concentrated solution that contains the necessary components for denaturing and solubilizing proteins prior to their separation by size on a polyacrylamide gel.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g slurry, by Santa Cruz Biotechnology (3 mentions)
Np 40 lysis buffer, by Beyotime (3 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Anti flag agarose gel, by Merck Group (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Protein a agarose bead, by Thermo Fisher Scientific (2 mentions)
P1005, by Beyotime (1 mentions)
Twist, by Abcam (1 mentions)
E cadherin antibody, by Abcam (1 mentions)
Mta2 antibody, by Proteintech (1 mentions)
Twist antibody, by Abcam (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti human hax 1, by BD (1 mentions)
Bca kit, by Solarbio (1 mentions)
Cell lysis buffer, by Solarbio (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
14 protocols using sds loading buffer
1
Immunoprecipitation and Western Blotting
2
Flag Protein Immunoprecipitation Protocol
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Transiently transfected cells were washed twice with PBS and lysed in NP-40 lysis buffer (product number P0013F; Beyotime) supplemented with protease inhibitor (P1005; Beyotime). Whole-cell lysate was first precleared with protein A/G slurry (catalog number sc-2003; Santa Cruz). After centrifugation at 1,000 × g for 5 min at 4°C, supernatant along with 1 μg of mouse anti-Flag MAb and 50 μL of protein A/G slurry (catalog number sc-2003; Santa Cruz) was incubated with rotation for 4 h at 4°C. Immunoprecipitated samples collected by centrifugation were washed with NP-40 lysis buffer four times. The final pellet was dissolved in 4× SDS loading buffer (catalog number P1016; Solarbio) for SDS-PAGE and Western blotting.
3
Flag Protein Immunoprecipitation Protocol
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Transiently transfected cells were washed twice with PBS and lysed in NP-40 lysis buffer (product number P0013F; Beyotime) supplemented with protease inhibitor (P1005; Beyotime). Whole-cell lysate was first precleared with protein A/G slurry (catalog number sc-2003; Santa Cruz). After centrifugation at 1,000 × g for 5 min at 4°C, supernatant along with 1 μg of mouse anti-Flag MAb and 50 μL of protein A/G slurry (catalog number sc-2003; Santa Cruz) was incubated with rotation for 4 h at 4°C. Immunoprecipitated samples collected by centrifugation were washed with NP-40 lysis buffer four times. The final pellet was dissolved in 4× SDS loading buffer (catalog number P1016; Solarbio) for SDS-PAGE and Western blotting.
4
Immunoprecipitation and Western Blot Analysis
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For immunoprecipitation experiments, whole-cell lysates were prepared as the method of immunoblot assay, followed by incubation with the anti-Flag agarose gels (Sigma) overnight at 4 °C. The beads were washed five times with low-salt lysis buffer, and then resuspended with 2 × SDS Loading Buffer (Solarbio), and boiled for 10 min. The released proteins were subjected to western blot analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were incubated with indicated antibodies overnight at 4 °C, and added with 20 μl Dynabeads protein G (Invitrogen) for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were incubated with indicated antibodies overnight at 4 °C, and added with 20 μl Dynabeads protein G (Invitrogen) for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
5
Protein-Protein Interaction Detection
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To detect the protein–protein interaction, the immunoprecipitation experiments were performed. Whole cell lysates were prepared as the method of immunoblot assay, and incubated with the anti-Flag agarose gels (Sigma) on roller shaker overnight at 4 °C. The beads were washed six times with low salt lysis buffer, re-suspended with 2× SDS Loading Buffer (Solarbio), and boiled for 10 min. The released proteins were subjected to western blot analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were obtained as the method of immunoblot assay, and then incubated with indicated antibodies overnight at 4 °C. Twenty microliter Dynabeads protein G (Invitrogen) were added to the extracted cell proteins and incubated for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were obtained as the method of immunoblot assay, and then incubated with indicated antibodies overnight at 4 °C. Twenty microliter Dynabeads protein G (Invitrogen) were added to the extracted cell proteins and incubated for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
6
Coimmunoprecipitation of Acetylated Proteins
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Cells were harvested 72 h after transfection, and nuclear protein was extracted using NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) following the manufacturer's protocol. A small amount of nuclear protein lysate was prepared for western blotting, and the remaining lysate was incubated overnight at 4°C with MTA2 antibody (Proteintech) and acetylated‐lysine antibody (CST), and rabbit IgG antibody as a negative control. Next, the lysates were pre‐cleared with prepared protein A‐agarose beads (Thermo Fisher) for 2 h at 4°C with gentle agitation. After high‐speed centrifugation to remove the supernatant, the beads were washed 3 times with immunoprecipitation washing buffer and then boiled for 5 min in 2× SDS loading buffer (Solarbio). After centrifugation, the supernatant was used for western blot analysis. Membranes were incubated with Twist (Abcam) and E‐cadherin antibody.
7
Nuclear Protein Extraction and Immunoprecipitation
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Cells were harvested 72 h after transfection, and nuclear protein was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, USA) following the manufacturer's protocol. A small amount of nuclear protein lysate was prepared for western blotting, and the remaining lysate was incubated overnight at 4 °C with an MTA2 antibody, and rabbit IgG antibody as a negative control. Afterwards, the lysates were precleared with prepared protein A-agarose beads (Thermo Fisher, USA) for 2 h at 4 °C with gentle agitation. After a high-speed centrifugation to remove the supernatant, the beads were washed three times with immunoprecipitation washing buffer and then boiled for 5 min in 2 × SDS loading buffer (Solarbio, China). After centrifugation, the supernatant was used for western blot analysis. The membranes were incubated with a Twist antibody (Abcam, UK).
8
Western Blot Analysis of Protein Expression
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Cells were collected and lysed in cell lysis buffer (Solarbio) for 30 min on ice and then centrifuged at 12,000 rpm (15 min, 4 °C). The supernatant was collected and the concentration of total protein was determined with a bicinchoninic acid protein assay (BCA)Kit (Solarbio). The samples were mixed with 5× SDS loading buffer (Solarbio) and boiled at 100 °C for 10 min. An equal amount of each sample was then separated by SDS–PAGE and the proteins were transferred onto a polyvinylidene difluoride membrane. The membranes were then incubated with 5% BSA at room temperature for 1–2 h, followed by primary antibodies (mouse-anti-human HAX-1 from BD; from Proteintech; Rabbit anti-mouse NLRP3, ASC, Caspase1, Cleaved Caspase1, GasderminD, Cleaved GasderminD, IKKε, pIKKε, TBK1 and pTBK1 from CST; β-actin from Bioworld Technology) incubation at 4 °C for 12 h. Membranes were then washed three times with TBST and incubated with HRP-conjugated secondary antibodies (ZSGF-BIO). Visualization was achieved using a SuperSignal West Pico Trial Kit (Thermo). Relative intensities of the bands were analyzed by Image-J software. Three individual biological replicates were performed in a Western blotting assay.
9
Western Blot Analysis of Apoptosis-Related Proteins
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Eyeball tissues were ground to powder in a mortar with addition of liquid nitrogen. The tissues were lysed using RIPA lysate (Beyotime, China) on ice for 0.5 h and transferred to an EP tube. Then, the samples were ultrasonicated for 3 min and centrifuged at 12 000 g at 4°C for 20 min. The supernatant samples were then moved in another EP tube. Using BCA kits (Beyotime), the protein concentrations were quantified. The samples were added to 5×SDS loading buffer (#S8010; Solarbio, China) and boiled at 100°C for 5 min. Afterwards, the sample was separated via 8% SDS-PAGE and transferred onto PVDF membranes for 1.5 h. The membrane was sealed by 5% milk/TBST for 1 h. Primary antibody was diluted with 1% BSA/PBST according to the recommended dilution ratio. The membrane was incubated by primary antibody against cleaved Caspase-3 (1: 1000; #9664T; CST, USA), Caspase-3 (1: 1000; #9662S; CST), p53 (1: 1000; #60283-2-lg; Proteintech, Wuhan, China), Bcl-2 (1: 1000; #26593-1-AP; Proteintech) and Bax (1: 1000; #60267-1-AP; Proteintech) at 4°C overnight and incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1: 5000; #SA00001-2; Proteintech) at room temperature for 1 h. The protein bands were developed and analyzed using the ChemiDoc™XRS+ Gel imaging system (Bio-Rad, USA).
10
Immunoblotting Analysis of Transfected Proteins
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The indicated plasmids were co-transfected into HEK-293 T cells. At 24 h post-transfection, the cells were lysed with RIPA buffer. Total cell protein was collected and boiled for 10 min, 5 × SDS loading buffer (Solarbio) was added, and the samples were centrifuged (10 000 × g, 10 min, 4 °C). Then, each sample was subjected to SDS–PAGE followed by transfer onto PVDF membranes (Merck Millipore). The membranes were blocked with 5% BSA (Solarbio) for 2 h at RT and then incubated with a specific primary antibody at 4 °C for 12 h with shaking. After being washed with PBST (0.5% Tween 20), the membranes were incubated with secondary antibodies for 2 h at RT. After being washed using PBST, protein bands were observed and imaged by an Amersham Imager 600 RGB (GE, Marlborough, USA). The following antibodies were used: mouse anti-HA-HRP (1:1000 dilution), mouse anti-Flag-HRP (1:1000 dilution), mouse anti-Myc-HRP (1:2000 dilution), mouse anti-GAPDH (1:3000 dilution), rabbit anti-IRF3 (1:1000 dilution), rabbit anti-TBK1 (1:1000 dilution), phospho-TBK1 (Ser172) (1:1000 dilution), and phospho-IRF3 (Ser386) (1:1000 dilution).
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