Protein from cells and tissue was extracted using Passive Lysis Buffer (Promega) with protease/phosphatase inhibitor mini tablets, EDTA free (Pierce). Cells were resuspended and rocked in lysis buffer for 15 minutes, centrifuged at maximum speed for 1 minute. Supernatants were then collected. Frozen tissue was homogenized in lysis buffer using Bullet Blender Bead Lysis tubes (MidSci) and a Bullet Blender Storm 24 (Next Advance) set at Speed 10 for approximately 10 minutes. Homogenates were centrifuged at 4°C at maximum speed for 1 minute, and supernatants were collected. Protein concentration was determined using the BCA assay (Pierce). Cell and tissue protein extracts (35–40μg protein/lane) were loaded onto a 4–20% SDS-gel (BioRad). Proteins were transferred onto PVDF membrane (Millipore). Membranes were incubated in blocking solution (5% milk in TBST). Membranes were incubated with primary antibodies: THM1 (Sigma), phosphorylated AKT (Cell Signaling), phosphorylated ERK (Cell Signaling), AKT (Cell Signaling), ERK (Cell Signaling), ß-Actin (Cell Signaling), PPARγ (Cell Signaling), and CEBPα (Cell Signaling) overnight at 4°C. Membranes were washed and incubated with secondary antibodies conjugated to HRP (Cell Signaling). Signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and imaged using an Amersham Imager 600.
Sds gel
Manufactured by Bio-Rad
Sourced in United States
The 10% SDS gel is a laboratory equipment product that is used for the separation and analysis of proteins based on their molecular weight. It is a type of polyacrylamide gel electrophoresis (PAGE) system that utilizes the anionic detergent sodium dodecyl sulfate (SDS) to denature and coat proteins with a uniform negative charge, allowing them to be separated efficiently according to their size.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose filter, by Thermo Fisher Scientific (2 mentions)
β actin, by Abcam (2 mentions)
Amersham ecl plus western blotting reagents, by GE Healthcare (2 mentions)
Amersham ecl, by Cytiva (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Super signal west femto ultra sensitive enhanced chemiluminescent ecl substrate, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid protein assay, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions)
Imager 600, by Cytiva (1 mentions)
Bullet blender storm 24, by Next Advance (1 mentions)
Phosphorylated akt, by Cell Signaling Technology (1 mentions)
Bullet blender bead lysis tubes, by MidSci (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
31 protocols using sds gel
1
Protein Extraction and Western Blot Analysis
2
Retinal Protein Analysis by Western Blot
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Retinas were homogenized in lysis buffer. Equal amounts of protein samples were loaded in each well of a 4-20% SDS gel (Bio-Rad) and standard WB procedures were performed with HRP-conjugated secondary antibodies (Bio-Rad); signals were visualized on x-ray film using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and quantified using ImageJ software (NIH); β-actin was used as a loading control. Primary antibodies are listed in supplementary material Table S1 .
3
Exosome Protein Extraction and Analysis
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RIPA buffer with 1% protease inhibitor was prepared for lysis of captured exosomes. 30 µL of the prepared buffer solution was injected to postcapture ExoBeads and incubated for 20 min. After incubation, the protein lysate was aspirated and stored separately. Total amount of proteins in the lysate from the ExoBeads was measured by standard micro bicinchoninic acid (BCA) analysis according to the manufacturer's instructions. Western blot analysis was performed on a precast 4–20% SDS gel (BioRad, USA). The samples were prepared in 4× Laemelli buffer with 2‐mercaptoethanol and heated to 90 °C for 6 min before loading onto the gel. The gel was run at 120 V for 50 min before transferring at 120 V for 1 h on ice. Blocking was performed in 5% nonfat milk in tris‐buffered saline with 0.1% Tween® 20 detergent (TBST) for 90 min. Primary antibody incubated overnight on a rocker at 4 °C at a concentration of 1:1000 in 3% nonfat milk in TBST. Thorough rinsing was performed, and then secondary antibody was incubated for 90 min at room temperature at 1:1500 in 3% nonfat milk in TBST.
4
Profiling PRL-3 and ERK Signaling
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Cells were lysed using Qproteome lysis buffer (Qiagen, Cat # 37901), then spun at 14,000 rpm for 10 min at 4 °C. Protein concentration in the supernatant was quantified using BCA assay (Thermo Scientific, Cat # 23227). 30 μg of protein was loaded into each lane of a TGX-stain-free pre-cast 4–20% SDS gel (Biorad, Cat # 4568094), total protein was quantified upon stain-free gel imaging, and protein was transferred onto PVDF membrane using the Trans-Blot Turbo Transfer System (BioRad, Cat # 1704150). Membranes were blocked with 5% milk in 1% TBST for 1 h, and a 1:1000 dilution of Anti-PRL-3 antibody (R&D Biosystems, Cat # MAB3219), 1:5000 phospho-ERK (Cell Signaling, Cat # 19101) or 1:5000 total ERK (Cell Signaling, Cat # 137F5) was added overnight. Following three washes in TBST, secondary HRP-conjugated antibody (GE, Cat # NA9340V) were added at a 1:5000 dilution for 1 h and membranes imaged using Clarity Western ECL Substrate. (BioRad, Cat # 1705061).
5
Protein Extraction and Western Blot Analysis of THM2
6
Quantifying Protein Binding on NPs
7
Spinal Cord Protein Expression Analysis
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Selected gene expression changes, assessed by qPCR, were evaluated at the protein level using western blotting. Four slides of 50 µm thick cryosections, 5 sections per slide, from 4 of the young, middle and old, fresh-frozen spinal cords (randomly selected from cords processed in Section 2.1)
were taken equidistant along the rostro-caudal axis of the cervical spinal cord and proteins extracted in RIPA buffer. After brief sonication, samples were centrifuged for 15 min at 4ºC and supernatants collected and stored at -80ºC. 30-50 µg of protein was heated to 95ºC for 5 min in Laemelli loading buffer and electrophoresed in a 4-20% SDS gel (BioRad). Proteins were transferred to nitrocellulose membranes for 60 min at 100V at 4ºC. Membranes were blocked in 5% BSA or non-fat milk for 1 hour at room temperature and incubated with primary antibodies (1:500 Rab anti Acat1 (Cayman Chemical); 1:1000 Rab anti Mbp (Sigma); 1:1000 Rab anti Cnp (Sigma); 1:1000 Rab anti Olig2 (Millipore); 1:5000 Mse anti Mog (Millipore); 1:10 000 Mse anti Gfap (Millipore); 1:400 Rab anti Iba1 (Wako)) at 4ºC for up to 72 hrs. Secondary antibodies were applied for 1.5 hrs at room temperature and protein bands visualized using enhanced chemiluminescence. Protein was normalized to β-Actin (1:10 000, GE Healthcare Life Sciences) and bands were analyzed using Image J.
were taken equidistant along the rostro-caudal axis of the cervical spinal cord and proteins extracted in RIPA buffer. After brief sonication, samples were centrifuged for 15 min at 4ºC and supernatants collected and stored at -80ºC. 30-50 µg of protein was heated to 95ºC for 5 min in Laemelli loading buffer and electrophoresed in a 4-20% SDS gel (BioRad). Proteins were transferred to nitrocellulose membranes for 60 min at 100V at 4ºC. Membranes were blocked in 5% BSA or non-fat milk for 1 hour at room temperature and incubated with primary antibodies (1:500 Rab anti Acat1 (Cayman Chemical); 1:1000 Rab anti Mbp (Sigma); 1:1000 Rab anti Cnp (Sigma); 1:1000 Rab anti Olig2 (Millipore); 1:5000 Mse anti Mog (Millipore); 1:10 000 Mse anti Gfap (Millipore); 1:400 Rab anti Iba1 (Wako)) at 4ºC for up to 72 hrs. Secondary antibodies were applied for 1.5 hrs at room temperature and protein bands visualized using enhanced chemiluminescence. Protein was normalized to β-Actin (1:10 000, GE Healthcare Life Sciences) and bands were analyzed using Image J.
8
Western Blot Analysis of P-glycoprotein
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Cell lysates were extracted using Triton X-100 lysing buffer, and lysate was quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA, 23225). For each cell lysate, 20 μg of protein was run on a 4–15% SDS gel (Bio-Rad, Hercules, CA, USA, 4561083DC) and electrotransferred onto a PVDF membrane. The membrane was washed with TBS and blocked overnight with 3% BSA in TBST. The membrane was incubated with primary Rabbit anti-P-gp antibody (Abcam, Waltham, MA, USA, ab170904) for 2 h, washed 2× for 10 min with TBST, and incubated with a secondary Goat anti-Rabbit IgG HandL (Abcam, Waltham, MA, USA, ab97051) antibody for 1 h. Rabbit anti-β-actin antibody (Abcam, Waltham, MA, USA, ab8227) along with the MDR AT3B-1 cell line were used as controls (Figure 3 B). As per manufacturer’s instructions, the primary antibody detects the predominant protein band migrating in the region of 180–200 kDa and typically will demonstrate a smear on the membrane in the region of 150–300 kDa due to the glycosylation profile of the protein [66 (link)].
9
FGFR1 Western Blot Analysis
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Cells were lysed in RIPA buffer and loaded on 4%‐15% SDS gel (Bio‐Rad Laboratories, Inc). Gels were blotted on nitrocellulose membranes (Trans‐Blot Bio‐Rad Laboratories, Inc) and then incubated with anti‐FGFR1 antibody (D8E4, #9740, Cell Signaling). Signals were developed using Western Plus‐ECL (PerkinElmer). Expression of PARK7 (ab18257, Abcam) was used as a loading control.28
10
Protein Immunoblotting Assay Protocol
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For preparation of protein lysates, animals were washed from plates and boiled in 4% SDS loading buffer at 95°C for 2 min. Lysates were separated on a 4–15% SDS gel (BIO-RAD), transferred to an Immobilon PVDF membrane (EMD Millipore) and probed with the following antibodies: monoclonal mouse anti-Flag antibody (Sigma-Aldrich, F1804) and monoclonal mouse anti-α-tubulin antibody (Sigma-Aldrich, T6199) diluted 1:2000 in blocking solution overnight. Following the primary incubation, blots were incubated with goat anti-mouse-IgG horseradish peroxidase-conjugated secondary antibody (MilliporeSigma, 12-349), diluted 1:5000 in blocking solution for 1 h. Immunoblots were then developed using an ECL kit (Thermo Fisher Scientific) and X-ray film (Phenix).
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