Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher, USA). Protein concentration was measured using Bio-Rad protein assay reagent (Bio-Rad, USA). Equal amounts of proteins (40 μg) of each sample were separated by 10% sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). PVDF membrane was blocked in skim milk at 25°C for 2 h, and subsequently incubated at 4°C overnight with antibodies against AKT-2, Bcl-2, Bax, active caspase-9, active caspase-3 or GAPDH (all in 1 : 1000 dilution, Santa Cruz, USA) in Tris-buffered saline with Tween-20 (TBST) containing 5% defatted milk. Next, PVDF membrane was incubated with corresponding horseradish peroxidase-conjugated (HRP)-linked secondary IgG antibodies (anti-mouse or anti-rabbit, 1 : 1000 dilution, Santa Cruz, USA) for 1 h at room temperature. The bands of bound secondary antibody were detected with an enhanced chemiluminescence kit (Pierce Biotechnology, USA) and the signals were detected with a SuperSignal Protein Detection kit (Pierce Biotechnology, USA). The band intensity of western blot was quantified subsequent to normalization with the density of GAPDH using ImageJ (National Institutes of Health, USA).
Pvdf membrane
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom, China
PVDF membranes are a type of laboratory equipment used for various applications in life sciences. They are made from polyvinylidene fluoride, a durable and chemically resistant polymer. PVDF membranes are commonly used for protein transfer and detection in Western blotting techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (19 mentions)
Pvdf membrane, by Bio-Rad (8 mentions)
Pvdf membrane, by Thermo Fisher Scientific (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (3 mentions)
Sds page, by Bio-Rad (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Criterion polyacrylamide gel, by Bio-Rad (3 mentions)
Actin clone c4, by Merck Group (3 mentions)
Grb2 clone 23, by Santa Cruz Biotechnology (3 mentions)
Gangnam stain, by iNtRON Biotechnology (2 mentions)
Semi dry transblot, by Bio-Rad (2 mentions)
Bca protein assay kit, by Santa Cruz Biotechnology (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Cellytic m cell lysis reagent, by Merck Group (2 mentions)
Goat anti rabbit 800cw, by LI COR (2 mentions)
Goat anti mouse 680rd, by LI COR (2 mentions)
63 protocols using pvdf membrane
1
Western Blot Analysis of Apoptosis Markers
2
Verifying Protein Antibody Specificity
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Western blotting for BRCA2, XPD and APE1 proteins was performed in order to verify the specificity of the antibodies used for the respective proteins, as previously described [13 (link)]. For this, immunoprecipitated BRCA2, XPD and APE1 proteins were blotted onto PVDF membrane (Santa Cruz Biotechnology, Inc., USA, 0.45 μm pore size) using Mini PROTEAN Tetra Cell (Bio-Rad, USA).
3
Western Blot Analysis of Protein Expression
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Protein samples’ loading was done in order to perform 12–15% SDS PAGE. For the identification of the molecular weight of proteins, a pre-stained protein marker (Gang Nam-STAIN™, iNtRon Biotechnology, Inc., Seongnam, Korea) was utilized as a guide during the study. This protein marker included a wider range of molecular weights i.e. 10 –245 kDa. Then, transfer of proteins to PVDF membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was done through Semi Dry Trans-Blot (Bio Rad). Next to it, primary antibodies including HO-1, Nrf-2, PSD95, SYP, p-JNK and β-actin (Table 1 ) were applied at 4 °C overnight for the detection of various proteins after which, 5% (w/v) skim milk for blocking the membranes from the attachment of non-specific proteins was used. It was followed by rinsing the blots and incubating them at room temperature with anti-mouse secondary antibody for 2 h. Visualization of membranes was done using ECL and the achieved results were developed on X-ray films that were scanned later.Table 1
List of primary and secondary antibodies with catalog numbers.
| S.# | Antibodies Name | Catalogue # |
|---|---|---|
| 1 | Anti–HO–1 | sc-136960 |
| 2 | Anti-Nrf-2 | sc-365949 |
| 3 | Anti-PSD95 | sc-71933 |
| 4 | Anti-SYP | sc-17750 |
| 5 | Anti-p-JNK | sc-6254 |
| 6 | Anti-β actin | sc-47778 |
| 7 | Goat anti-mouse (IgG-HRPs) secondary antibodies | W4028 |
4
Testis Protein Extraction and Western Blot
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Total testis or enriched cells fractions were lysed in ice-cold protein extraction buffer containing 0.1% Nonidet P-40, 50 mM Tris–HCl, pH 7.9, 150 mM NaCl, 3 mM MgCl2, 3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF and protease inhibitors (ThermoFisher Scientific, A32965) followed by sonication (3 pulses of 10 s) using micro ultrasonic cell disrupter (Kontes). The relative amount of protein was determined measuring absorbance at 260 nm using NanoDrop 2000c spectrophotometer (ThermoFisher Scientific). Proteins were solubilized with 2× sample buffer (4% SDS, 160 mM Tris–HCl, pH 6.8, 20% glycerol, 4% mM β-mercaptoethanol, and 0.005% bromophenol blue) and 30 µg/lane of sample were separated by 4–15% gradient Tris–acetate SDS–PAGE and electro transferred to PVDF membrane (Santa Cruz Biotechnology, sc-3723). The blots were probed with individual primary antibodies, and then incubated with HRP-conjugated goat anti-mouse or rabbit antibody as required. In all blots, proteins were visualized by enhanced chemiluminescence, and images acquired using Western Blot Imaging System c600 (Azure Biosystems). ImageJ software were used for quantification of non-saturated bands and α-tubulin were used for normalization. Antibodies used are detailed in Additional file 11 : Table S4.
5
Immunoblotting of Notch1 and Dll4
6
Quantifying Protein Levels in Mouse Brains
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Western blotting was carried out according to the method reported by Badshah et al. [44 (link)] with minor alterations. The mice brains were collected as quickly as possible and then homogenized in a T-PER solution. The levels of proteins in all 4 groups were measured using a BioRad protein assay. For each of the 4 groups, 30 μg of the protein content was run on a 15–20% SDS-PAGE. After completing the electrophoresis process, all the proteins were shifted to a PVDF membrane (Santa Cruz Biotech, USA) over transblot (Bio-Rad). Different primary monoclonal antibodies (Santa Cruz, CA, USA) such as caspase-3, Bcl-2-associated X protein (BAX), beta-secretase-1 (BACE-1), poly (ADP-ribose) polymerase-1 (PARP-1), amyloid beta (Aβ), synaptophysin (SYP), postsynaptic density protein 95 (PSD-95), and beta actin as well as HRP-conjugated secondary antibodies (Santa Cruz, CA, USA) were smeared. All results were visualised in a dark room on X-ray films.
7
BRCA2, XPD, and APE1 Protein Assay in HNSCC
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The assay for total BRCA2, XPD and APE1 protein of PBL from 100 HNSCC patients and 122 controls was performed by slot blot immunoprobe assay as described earlier [13 (link)]. Slot blotting technique is used for screening a large number of samples as it is quick, efficient and requires less amount of sample [13 (link)]. For this, 500 ng of total protein was blotted in triplicate onto PVDF membrane (Santa Cruz Biotechnology, Inc., USA, 0.45 μm pore size) using Bio-Dot SF microfiltration apparatus (Bio-Rad, USA).
8
Western Blot Analysis of Apoptosis Regulators
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Proteins were extracted with CelLyticTM M Cell Lysis Reagent (Sigma‐Aldrich, Dorset, UK) supplied with protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, Dorset, UK). Proteins were mixed with NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min before analysis by western blot analysis. Proteins were subjected to 4–20% NuPAGE gels (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Sigma‐Aldrich, Dorset, UK) at 20 V for 1 hr by semidry transfer. PVDF membrane was blocked with 5% nonfat milk in TBST for 1 hr and then incubated with primary antibodies overnight at 4°C against the following targets: Bcl‐2 (100), RelA (NF‐κB p65, F‐6), Bcl‐XL (H‐5), Bax (2D2), Survivin (D‐8), and LDH (H‐10) (Santa Cruz Biotechnology, Inc., Dallas, TX); β‐actin (AC‐74) (Sigma‐Aldrich, Dorset, UK); p‐RelA (phospho‐NF‐κB p65‐Ser536), p‐STAT3 (phosphor‐STAT3‐Tyr705), p‐AKT, AKT, Mcl‐1, and Rb antibodies (Cell Signaling Technology‐New England Biolabs, Hitchin, UK). Bound antibodies were detected using appropriate horseshoeradish peroxidase‐conjugated secondary antibodies and visualized by GeneSnap (SynGene, Cambridge, UK) after adding ECL plus (GE Healthcare Life Science, Hatfield, UK).
9
Reversing Immortalization in iDP6 Cells
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Primary DP cells and iDP6 cells were cultured. The iDP6 cells were treated with AdGFP (adenovirus with the ability to express GFP protein), AdFlip (adenovirus with the ability to express flip recombinase, which can interact with FRT thus remove the expression of SV40) or PBS. Forty-eight hours later, cells were collected and total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China). Then, total proteins were loaded to 1% SDS-PAGE gel (Beyotime, China) and transmitted to PVDF membrane (Bio-Rad, Hercules, CA, USA). The PVDF membrane were incubated with anti-SV40 (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-GAPDH (1:500; ZSGB-bio, Beijing, China) antibodies. HRP labelled secondary antibodies were used, and the results were observed under ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA). The experiment on reversing immortalization was performed twice.
10
Western Blot Protein Analysis Protocol
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The western blot procedure was carried out as previously described by Farooq et al., (2021) (link). All brain tissues were homogenized, centrifuged and then loaded to perform 12–15% SDS PAGE. To record the desired protein's molecular weight; a pre-stained protein marker (Gang Nam-STAIN™, iNtRon Biotechnology, Inc., Seongnam, Korea) that covers a broad range of molecular weights, i.e., 10–245 kDa range was used throughout the study. It was followed by the transfer of proteins to PVDF membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using Semi Dry Trans-Blot (Bio-Rad). A mouse derived primary monoclonal antibodies were applied at 4 °C overnight (1:1000 in TBST) to detect different targeted proteins (HO-1, COX-2, Nrf-2, TNF-α, NF-κB, and β-actin). The binding of non-specific proteins was reduced by using 5% (w/v) skim milk which blocked the membranes. The blots were rinsed, and then incubated for two hours at room temperature with horseradish-peroxidase-conjugated goat anti-mouse (IgG-HRPs) secondary antibody (1:2000 in TBST). The bands of the western blot were visualized using enchanced ochemiluminescence solution, a detection reagent according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). Results obtained were developed on the X-ray films, which were later scanned.
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