Total protein from proximal and distal IPA samples (n=8/each) was extracted using RIPA buffer (150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris at a pH of 7.65). Protein concentration was determined through bicinchoninic acid protein assay and 20 μg of protein was loaded in 4–12% Bis-Tris SDS-polyacrylamide gel (Thermo Fisher, Waltham, MA, USA). Protein electrophoresis at 160 V for 60 minutes was conducted and proteins were transferred to nitrocellulose membrane. Membranes were incubated with rabbit polyclonal primary antibodies against either P2X1 (1:1,000) or P2Y1 (1:200) receptors (Alomone Labs, Jerusalem, Israel) and conjugated with goat anti-rabbit polyclonal horseradish peroxidase antibody (1:5,000; Thermo Fisher, Waltham, MA, USA). Bands were detected by chemiluminescence (Pierce ECL) and imaged with Bio-Rad ChemiDoc Touch (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were stripped and ß-actin (1:1,000; Thermo Fisher) expression was evaluated to normalize P2X1 and P2Y1 receptor protein expression. Densitometry of the bands were analyzed using ImageJ software (NIH, Bethesda, MD, USA).
Bis tris sds polyacrylamide gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bis-Tris SDS-polyacrylamide gel is a laboratory equipment used for protein separation and analysis. It utilizes a Bis-Tris buffer system and sodium dodecyl sulfate (SDS) to separate proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Chemidoc touch, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Searchlight, by Thermo Fisher Scientific (2 mentions)
Protran 0.45 μm nitrocellulose membrane, by GE Healthcare (1 mentions)
Goat anti mouse igg conjugated to irdye800cw, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Pierce tmb subs trate kit, by Thomas Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (1 mentions)
Phosphorimaging, by GE Healthcare (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Peroxidase affinipure goat anti mouse igg and goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ab32136, by Abcam (1 mentions)
A2066, by Merck Group (1 mentions)
P8340, by Merck Group (1 mentions)
11 protocols using bis tris sds polyacrylamide gel
1
Quantification of P2X1 and P2Y1 Receptors
2
Quantifying Mitochondrial Respiratory Complexes
3
Quantification of PfCRT Protein in Oocyte Membranes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The preparation of oocyte membranes and the semi-quantification of PfCRT protein was carried out using a protocol described in detail elsewhere [42 (link)]. Protein samples prepared from oocyte membranes were separated on a 4–12% Bis-Tris SDS-polyacrylamide gel (Life Technologies) and transferred to a Protran 0.45 μM nitrocellulose membrane (Amersham, GE Healthcare Life Sciences). The membranes were probed with rabbit anti-PfCRT antibody (concentration of 1:4,000; Genscript) followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (1:8,000; Life Technologies, cat. no. 656120). Validation of the specificity of the anti-PfCRT antibody has been published in detail elsewhere [42 (link)]. The PfCRT band for each variant was detected by chemiluminescence (Pierce), quantified using the Image J software [75 (link)], and expressed as a percentage of the intensity measured for the PfCRTK1 band. Total protein staining was used to evaluate sample loading and efficiency of transfer as outlined previously [42 (link)]. Between five and seven independent experiments were performed (on oocytes from different frogs), and in each experiment measurements were averaged from two independent replicates.
4
SDS-PAGE Analysis of LVS Lysate
5
Western Blot Protein Quantification
6
Assessing Protein-DNA Adduct Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reactions were incubated in 25 mM NaPO4 pH 7.4, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 125 mM KCl and 1 mM dithiothreitol at 37°C and terminated by the addition of 200 mM NaBH4 and an additional incubation on ice for 1 h. The products were analyzed by denaturing polyacrylamide gel electrophoresis and phosphorimaging (GE Biosciences). For velocity determination, aliquots were taken at three different times from triplicate reactions, with the duration of sampling increased for mutant proteins with very low activity to ensure the fraction of substrate that was converted to product was in a range that was accurately quantifiable (greater than 1%).
The covalent protein-DNA adducts were generated by assembly of reactions as above without Ku, and initiated by sequential addition of Ku and 5 mM NaBH4. Products were separated on a 4–12% Bis-Tris SDS polyacrylamide gel (Invitrogen) and analyzed by phosphorimaging (GE Biosciences).
EMSA analysis was performed by incubating 1 nM substrate with 1nM Ku for 15 min at 37°C, followed by electrophoresis on a 4% native polyacrylamide gel containing 1/3× TRIS/borate/EDTA buffer and analyzed by phosphorimaging (GE Biosciences).
The covalent protein-DNA adducts were generated by assembly of reactions as above without Ku, and initiated by sequential addition of Ku and 5 mM NaBH4. Products were separated on a 4–12% Bis-Tris SDS polyacrylamide gel (Invitrogen) and analyzed by phosphorimaging (GE Biosciences).
EMSA analysis was performed by incubating 1 nM substrate with 1nM Ku for 15 min at 37°C, followed by electrophoresis on a 4% native polyacrylamide gel containing 1/3× TRIS/borate/EDTA buffer and analyzed by phosphorimaging (GE Biosciences).
7
In vitro Neddylation Assay for DCNL1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DCNL1 co-E3 ligase activity was assessed in an in vitro neddylation assay with CUL5–RBX2 as substrates. Neddylation reactions containing 100 nm of either DCNL1, DCNL1 (PONY), DCNL1-UbCT, or DCNL1 (DADMUT), 25 nm NAE1, 50 nm N-terminal acetylated UBE2F (gift from Brenda Schulman and Danny Scott), 14 μm NEDD8, and 80 nm CUL5–RBX2 in reaction buffer (50 m HEPES-NaOH, pH 7.4, 50 mm NaCl, 5 mm MgCl2, 5% glycerol, 0.2 mm EDTA, 0.02% Triton X-100, and 5 mm Mg2+-ATP) were incubated at 37 °C and stopped after the indicated time points (0, 2, 5, and 10 min) by adding 4× SDS sample buffer complemented with DTT. 80% of the reaction was separated on 4–12% BisTris SDS-polyacrylamide gel (Invitrogen) for silver staining and 20% was analyzed by immunoblotting using anti-CUL5 antibody. Silver stained gels were scanned, NEDD8–CUL5 and CUL5 bands quantified using ImageJ software.
8
Liver Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver samples were homogenized in HEN buffer (10 mM HEPES pH 7.4, 10 mM EDTA pH 8.5, 25 mM NaCl, 1 mM PMSF and 1× complete protease inhibitor cocktail). 1% Triton X-100 was added and supernatant was removed and stored at −80 °C until ready to use. Samples were run on a 4–12% Bis-Tris SDS-polyacrylamide gel (Invitrogen) and transferred to a PVDF membrane overnight. The membrane was blocked in 5% nonfat dry milk (NFDM) in DPBS for 2 h at room temperature and probed with either rabbit-anti-mouse Albumin antibody diluted 1:1000 (Cell Signaling, Catalog #4929) or Gapdh antibody diluted 1:1000 (Cell Signaling, Catalog #2118) in 5% NFDM in DPBS with 0.1% Tween-20 (PBST) overnight at 4 °C. The membrane was then washed in PBST 3 times and place in horseradish peroxidase conjugated secondary antibody diluted 1:10,000 (Prometheus protein biology products, Catalog #20-304D) in 5% NFDM in DPBS at room temperature for 2.5 h. The membrane was then detected by chemiluminescence using a ChemiDoc Touch Imaging System (BioRad) for a visualization.
9
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as detailed elsewhere.22 (link),33 (link) Twenty micrograms of cellular proteins was electrophoresed through 10% Bis-Tris SDS-polyacrylamide gels (Thermo Fisher Scientific) and then transferred to a polyvinylidene difluoride membrane. The primary antibodies used for this experiment were obtained from Abcam (Cambridge, UK), including YARS2 (ab127542), ND1 (ab74257), CO2 (ab110258), TOM20 (ab56783), and total OXPHOS human WB antibody mixture (ab110411), and from Proteintech (Rosemont, IL, USA), including NDUFS1 (12444-1-AP), NDUFA9 (20312-1-AP), NDUFS3 (15066-1-AP), SDHA (14865-1-AP), UQCRC1 (21705-1-AP), COX5A (11448-1-AP), ATP5B (17247-1-AP), and VDAC (55259-1-AP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; AB-M-M 001) was obtained from Hangzhou Goodhere Biotechnology Co., Ltd. (Hangzhou, China), and light chain 3 (LC3; 4108) from Cell Signaling Technology (Danvers, MA, USA). Peroxidase AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG (The Jackson Laboratory, Bar Harbor, ME, USA) were used as a secondary antibody, and protein signals were detected using the ECL Western Blotting Analysis System (MilliporeSigma, Billerica, MA, USA). Quantification of density in each band was performed as detailed previously.22 (link),33 (link)
10
Platelet Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described by us (21 (link)). Platelet samples (−80°C) were thawed and tubes dissolved in 100 μL ice-cold PBS containing a protease inhibitor cocktail (P-8340; Sigma). Samples were then sonicated using an ultrasonic device, centrifuged at 14,000 × g for 10 min at 4°C; the extracts were denatured (10 min, 70°C), and 18 μg was loaded onto 10% bis–tris SDS-polyacrylamide gels (Thermo Fisher Scientific, Vienna, Austria), separated for 35 min at 200 V and finally electrotransferred to nylon-PVDF Immobilon-PSQ membranes for 20 min at 30 V in 20% methanol blotting buffer. Next, blots were blocked for 30 min in blocking buffer; incubated with primary antibody against APP (Abcam ab32136, 1:2,000, Cambridge, UK), or CD41 (Abcam ab63323, 1:2,000), or actin (1:1,000, A2066; Sigma, Vienna, Austria) at 4°C overnight; washed; and then incubated in alkaline phosphatase–conjugated anti–rabbit IgG for 30 min. After washing, bound antibodies were detected using an enhanced chemiluminescence system and visualized by using a cooled CCD camera (SearchLight; Thermo Fisher Scientific).
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!