Histone lysates were extracted using Histone Extraction Kit (Abcam, ab113476). Protein amounts were quantified using Micro BCA Protein Assay Kit (Thermo Scientific) Three microgram of histone lysates were separated on home-made 12% gels and transferred onto PVDF membranes (GE Healthcare) for 1 hour. Blocking was then performed using 5% Bovine Serum Albumin (BSA, Multicell) in Tris-buffered saline (50mM Tris, 150mM NaCl, pH 7.4, 0.1% Tween 20) (TBST) for one hour at room temperature. Membranes were incubated in the presence of the relevant antibody overnight at 4 degrees Celsius in 5% BSA in TBST: anti-H3K27M (1:200, Millipore ABE419) and anti-total H3 (1:2000, Abcam 1791). Membranes were washed three times with TBST prior to incubation with Horse Radish Peroxidase-linked secondary antibody (1:20000, Bethyl A120-100P) in 5% BSA in TBST for one hour at room temperature. Membranes underwent a final three-rounds of washes with TBST before the signal was developed with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Finally, signals were visualized using ChemiDoc MP Imaging System (Bio-Rad). Precision Plus Protein™ WesternC™ Blotting Standards (Bio-Rad, #1610376) was used as a molecular weight markers which covers weight ranges from 10 to 250kDa by 10 bands.
Precision plus protein westernc blotting standards
Manufactured by Bio-Rad
Sourced in United States
Precision Plus Protein™ WesternC™ Blotting Standards is a pre-stained molecular weight protein standard for use in Western blotting. It contains a mixture of ten recombinant proteins with molecular weights ranging from 10 to 250 kDa.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Histone extraction kit, by Abcam (1 mentions)
Abe419, by Abcam (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Clarity max western ecl substrate, by Bio-Rad (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Goat anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ab88443, by Abcam (1 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Ibright analysis software, by Thermo Fisher Scientific (1 mentions)
Npo335, by Thermo Fisher Scientific (1 mentions)
Deglycosylation enzyme cocktail, by New England Biolabs (1 mentions)
G7 reaction buffer, by New England Biolabs (1 mentions)
6 protocols using precision plus protein westernc blotting standards
1
Histone Lysate Western Blot Protocol
2
SDS-PAGE and Western Blot Analysis of Cystatin B
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AD and HC IPs were mixed 1:1 (v/v) with 60 mM Tris/HCl pH 6.8 containing 2% SDS, 20% glycerol, and 0.02% bromophenol blue with or without 2% 2-mercaptoethanol to perform SDS-PAGE under reducing (R) and non reducing (NR) conditions, respectively, using 4.5 μg of total protein for each IP. Electrophoretic separation was performed with the Mini-PROTEAN Tetra cell (Bio-Rad) at 180 V and Precision Plus Protein™ WesternC™ Blotting Standards (Bio-Rad) were used as molecular weight standards. Proteins were transferred to 0.2 μm PVDF membranes according to the manufacturer’s instructions for the Trans-blot Turbo system (Bio-Rad). After the transfer, PVDF membranes were equilibrated for 1 h with blocking solution (5% blotting-grade blocker, Bio-Rad, in TBS containing 0.05% Tween-20, TBS-T), and then for 1 h under stirring with cystatin B mouse monoclonal primary antibody (Invitrogen/Thermo-Fisher Scientific) diluted 1:1000 with TBS-T. After 5 × 5 min washing with TBS-T, membranes were incubated for 1 h with the anti-mouse secondary Ab (HRP conjugated dil. 1:5000 in TBS-T). After 5 × 5 min washing with TBS-T, membranes were incubated with the detection solution (Clarity Max™ Western ECL Substrate, Bio-Rad). The detection of cystatin B positive signals was performed with the ChemiDoc MP Imaging System (Bio-Rad) and analyzed with Image Lab 4.0.1 software.
3
Quantification of CYP1A2 and NAT2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression of CYP1A2 and NAT2 in yeast cells was determined by Western blot. Cells were inoculated in YPD (Original) or SC-URA medium (Containing pCYP1A2_NAT2) and grown to log phase (A600 between 0.5 and 1) and then concentrated. Protein extracts were prepared as described previously by Foiani et al. (1994) (link). Proteins were then separated on a 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. To detect human CYP1A2, a mouse anti-CYP1A2 antibody (1:1,000, Abcam: ab22717) was used, followed by a goat antimouse secondary antibody (1:10,000, Santa Cruz Biotechnology: sc-2005). To detect human NAT2, a polyclonal mouse anti-NAT2 antibody (1:1,000, Abcam: ab88443) was used, followed by the same goat antimouse secondary antibody (1:10,000, Santa Cruz Biotechnology: sc-2005). Precision Plus Protein Western C Blotting Standards (Bio-Rad) were used for molecular weight standards.
4
SDS-PAGE Analysis of EBN Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine protein distribution and the molecular weight of proteins in EBN and EBND, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the proteins in 2.5% (w/v) EBN and EBND. Briefly, 5 µL of EBN, EBND, digestive enzyme-only solution and protein standards (Precision Plus Protein™ WesternC™ Blotting Standards, 1610376, Bio-Rad, Hercules, CA, USA) were loaded into individual wells in a 12.5% polyacrylamide gel and ran under the constant current setting of 21 mA for 30 min with an electrophoresis apparatus (WSE-1010 cPAGE Ace, ATTO CO., Tokyo Japan). After electrophoresis, the gel was rinsed by DDW and stained with a quick CBB solution (299-50101, FUJIFILM Wako, Osaka, Japan) for 30 min. Clear bands were obtained by de-staining the gel with DDW. The protein gel was photographed with the iBright FL1500 Imaging System (Thermo Fisher Scientific, Tokyo, Japan). The protein bands were analyzed by iBright Analysis software (Thermo Fisher Scientific, Tokyo, Japan).
5
Western Blot Analysis of SIRPα
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum (diluted 1:50) and MDM culture supernatants were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (4–12% bis tris NuPAGETM Invitrogen Waltham, MA, USA catalogue number NPO335), according to the supplier’s instructions. Proteins were blotted onto PVDF membranes, blocked for 1 h, and incubated overnight at 4 °C with sheep anti-human SIRPα antibody (R&D Systems, catalogue number AF4546, 0.4 µg/mL for serum and 1.6 µg/mL for supernatants). The membrane was subsequently incubated for 1 h at room temperature (RT) with rabbit anti-sheep horseradish peroxidase, and developed using enhanced chemiluminescence. BioRad, Hercules, CA, USA, Precision Plus Protein™ WesternC™ Blotting Standards, catalogue no. #1610376, were used.
6
Deglycosylation of chIL-18BP Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
40 μl of FL or SV chIL-18BP concentrate was incubated with 5 μl 10× G7 reaction buffer (New England Biolabs (NEB), Ipswich, MA, USA) and 5 μl deglycosylation enzyme cocktail (P6039S, NEB) at 37 °C for 5 h. Deglycosylated proteins were resolved by SDS-PAGE and detected by Western blotting as described above. Precision plus protein™ WesternC™ blotting standards (BIO-RAD) were used to confirm the molecular weight of the applied recombinant proteins.
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!