An alkaline phosphatase assay kit (cat. no. A059-2) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Notch1 intracellular domain (N1ICD) and Jagged1 polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-rat GAPDH polyclonal antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. no. 2118). Jagged2 polyclonal antibody was purchased from Merck Millipore (Darmstadt, Germany; cat. no. NRG1764426). Bovine serum albumin was purchased from Roche Diagnostics (Basel, Switzerland). PrimeScript RT Reagent kit with gDNE Eraser (cat. no. AK3501) and RT-PCR SYBR® (cat. no. AK4401) kits were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Phenylmethanesulfonyl fluoride was purchased from Sigma-Aldrich (Merck Millipore). A bicinchoninic acid (BCA) protein assay kit was purchased from KeyGen Biotech. Co., Ltd. (Nanjing, China). An SDS-PAGE gel preparation kit, SDS-PAGE protein loading buffer (5X) and SDS-PAGE electrophoresis liquid were purchased from the Beyotime Institute of Biotechnology (Haimen, China). Goat anti-rabbit secondary antibody was purchased from EarthOx (Wuhan, China). Other commercially available reagents and chemicals used in the study were of analytical grade.
Sds page protein loading buffer 5x
Manufactured by Beyotime
Sourced in China
The SDS-PAGE protein loading buffer (5X) is a concentrated solution used to prepare protein samples for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The buffer contains the anionic detergent SDS, which denatures proteins and gives them a uniform negative charge, allowing for separation based on molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
Sds page gel preparation kit, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
Bicinchoninic acid bca protein assay kit, by Keygen Biotech (1 mentions)
Amersham hybond, by GE Healthcare (1 mentions)
Ripa tissue cell lysate, by Solarbio (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
Ha1024, by Huabio (1 mentions)
E cadherin, by Huabio (1 mentions)
N cadherin, by Huabio (1 mentions)
Vimentin, by Huabio (1 mentions)
Gapdh, by Huabio (1 mentions)
Ddit4, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Nvp bez235, by Selleck Chemicals (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
7 protocols using sds page protein loading buffer 5x
1
Alkaline Phosphatase Assay and Notch Pathway Protein Detection
2
Protein Extraction and Western Blot Analysis
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Eight tissue specimen pairs were cut into pieces and placed in 1% PMSF and RIPA tissue/cell lysate (Solarbio, China) on ice for 30 minutes. The lysed tissue was then centrifuged at 12,000 rpm at 4°C for 15 minutes. The supernatant was removed and the protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China). SDS-PAGE protein loading buffer (5X) (Beyotime, China) was added and the mixture was boiled for 10 minutes. The protein (30 μg) was added to a prepared 12% SDS-PAGE gel for electrophoretic separation and transferred to a 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare). The membrane was blocked with a rapid blocking solution for 30 minutes and incubated with EPHB2 (1:1000, AF5246, Affinity), SLC6A1 (1:1000, DF4510, Affinity), PPP1R17 (1:1000, PA5-61599, Invitrogen), PPARGC1A (1:1000, FNab06351, FineTest), or GAPDH (1:1000, ab181602; Abcam) antibodies overnight on a 4°C shaker and washed three times with TBST (0.1% Tween-20) for 10 minutes. The membrane was then incubated with goat anti-rabbit IgG (H + L) HRP (1:3000, S0001; affinity) for 1 hour, washed, and developed using BeyoECL Star (Beyotime, China).
3
Protein Extraction and Western Blot Analysis
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According to the instructions, PMSF and RIPA tissue / cell lysates (Solarbio, China) were added to cells or tissues, lysed on ice for 20 min, and then centrifuged for 15 min (4 ° C, 12,000 rpm). Then, the protein concentration of the supernatant was measured using the BCA concentration kit (Beyotime, China). Finally, SDS-PAGE protein loading buffer (5X) (Beyotime, China) was added, and then the protein was denatured by metal bath for 10 min. After the protein extraction was completed and the concentration was determined, an appropriate amount of sample protein was added to the electrophoresis gel for electrophoresis separation, and then transferred to the PVDF membrane (Solarbio, China). PVDF membranes were sealed with a rapid blocking solution and incubated overnight with primary antibody in a refrigerator at 4 ° C, and the next day with secondary antibody (1:20000, HA1024; HUABIO) were incubated at room temperature for 60 min. In the last step, we used the Ori Ultrasensitive ECL Kit (Oriscience, China) to observe the bands. The antibodies used were OLFM2 (1:1000, ab154196; Abcam), GAPDH (1:30000, SA30-01; HUABIO), E-cadherin (1:5000, ET1607-75; HUABIO), N-cadherin (1:1000,ET1607-37; HUABIO), Vimentin (1:20000,ET1610-39; HUABIO), TGF-βR1 (1:1000,PB1154; BOSTER), Phospho-Smad3 (1:1000,C25A9; CST), Phospho-Smad2 (1:1000, D27F4; CST).
4
Protein Extraction and Western Blot Analysis
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To obtain total protein, cells were subjected to protein extraction using 1% PMSF and RIPA buffer (Solarbio, Beijing, China, Cat No. R0020) on ice for 30 minutes. The resulting mixture was centrifuged at 12,000 rpm for 30 minutes, and the protein suspension was collected from the liquid supernatant. Protein concentration was determined using the BCA method (Epizyme, Shanghai, China, Cat No. ZJ101). Subsequently, SDS-PAGE protein loading buffer (5X) (Beyotime, China) was added to the protein suspension, followed by boiling for 10 minutes. The protein was then separated using either a 10% or 12.5% SDS-PAGE gel (Epizyme, Shanghai, China, Cat No. PG113 or PG112) and transferred onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane. To block the PVDF membranes, 5% skim milk was applied for 1.5 hours. Next, the membranes were incubated with primary antibodies including DDIT4 (ProteinTech, Wuhan, China, Cat No. 67059-1-Ig, 1:1000), BCL2 (ProteinTech, Wuhan, China, Cat No. 68103-1-Ig, 1:1000), Caspase-3 (Huaan, Hangzhou, China, Cat No. ET1602-39, 1:1000), and GAPDH (Huaan, Hangzhou, China, Cat No. ET1601-4, 1:5000), followed by incubation with corresponding secondary antibodies. Finally, the protein bands were visualized using chemiluminescence kits.
5
Inhibition of Akt and p70S6K Signaling in Cisplatin-Resistant Cells
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NVP-BEZ235 was purchased from Selleck Chemicals. DMSO was purchased from Aladdin Industrial Co., Ltd. (https://www.aladdin-e.com ) Cisplatin was purchased from Sigma-Aldrich (Merck KGaA). The Cell Counting Kit-8 (CCK-8) assay, BCA Protein Assay kit, Cell Cycle Analysis kit, Annexin V-FITC Apoptosis Detection kit, RIPA lysis buffer and SDS-PAGE protein-loading buffer (5X) were purchased from Beyotime Institute of Biotechnology. Giemsa stain was purchased from Shanghai Fushen Biotechnology Co., Ltd. The monoclonal Akt (cat. no. 4691S), phosphorylated (p)-Akt (cat. no. 2965S), p70-S6 Kinase (p70S6K; S6K; cat. no. 2708S), p-p70 S6 Kinase (p-p70S6K; p-S6K; cat. no. 9234S), ABCG2 (cat. no. 42078S), MRP1 (cat. no. 14685S) and β-actin (cat. no. 4970S) rabbit antibodies were purchased from Cell Signaling Technology, Inc. All primary antibodies were used at a dilution of 1:1,000. The HRP-labeled goat anti-rabbit IgG secondary antibody (cat. no. 111-035-003) was purchased from Jackson ImmunoResearch Laboratories, Inc. The secondary antibody was used at a dilution of 1:5,000.
6
Placental Protein Profiling in Normal and Pre-Eclampsia Pregnancies
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Proteins from placentas in women with normal pregnancy or PE (matched for gestational age, fetal sex and primiparity) and cell lines were extracted using a total protein extraction kit (Beyotime, China). The protein concentration was determined using a BCA protein quantification kit (Yeasen, China). All proteins were standardized to a concentration of 3 μg/μL and denatured at 100 °C for 5 minutes in SDS–PAGE protein loading buffer (5X) (Beyotime, China). The proteins were separated with a PAGE gel quick preparation kit (Yeasen, China) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Subsequently, the membranes were blocked with 5% skim milk powder (Solarbio, China) for 1 h and incubated with primary antibodies against CXCL3 (1:500, Sigma, USA), ERK1/2 (1:1000, ABclonal, China), p-ERK1/2 (1:1000, ABclonal, China) or β-actin (1:5000, ABclonal, China) overnight at 4 °C. Next, the membranes were washed three times with TBST and then incubated with a secondary antibody (1:100000, ABclonal, China) for 1 h at room temperature. Finally, SuperKine™ West Femto Maximum Sensitivity Substrate (Abbkine, China) was used to detect the chemiluminescence intensity with a ChemiDoc™ MP Imaging System (Bio-Rad, USA). Image Lab 6.0 software (Bio-Rad, USA) was used for gray value calculation. All experiments were conducted in triplicate.
7
Hippocampus Protein Extraction and Analysis
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Hippocampi were lysed using lysis buffer containing 20mM Tris, pH 7.5, 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,50mM NaF, 1mM Na3VO4 , 1mM PMSF, and a protease inhibitor ( 200612Signalway Antibody, USA). The BCA method was used to qualify protein concentration. The supernatant of hippocampus extract was mixed with SDS-PAGE protein loading buffer (5x) (Beyotime, China) in the ratio of 4:1 and then boiled. After SDS-PAGE, the proteins were transferred onto a polyvinylidene di uoride membrane ,blocked with 5% non-fat milk in TBST buffer (20mM Tris-HCl, pH 7.4, 137mM NaCl, and 0.1% Tween-20) for 1h at 37°C, and incubated overnight at 4°C with primary rabbit polyclonal antibodies against GAPDH (41549,1:3000, Signalway Antibody, USA),APP, BACE1, PS1 (38604, 24100, 33474, 1:1500, Signalway Antibody, USA) and PS2 (DF7809, 1:1000, A nity Biosciences, USA). After extensive wash, the membrane was incubated with the appropriate HRP-conjugated secondary antibody (L3012, 1:5000, Signalway Antibody, USA) and then developed using the Super ECL Plus reagents. The relative band intensities were quanti ed by densitometry using the ImageJ software (1.41V, US National Institutes of Health).
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