Primary antibody dilutions were prepared at 1:1000 to 1:500 in 5% BSA or dry milk in TBS with 0.05% v/v Tween. Protein samples prepared directly from lysate were prepped in 5X SDS sample buffer. Westerns were run in BioRad equipment in SDS buffer, using BioRad gradient gels, at 175 V for 42 min. Precision plus dual color protein marker (BioRad) was used in parallel to samples. Proteins were wet-transferred onto nitrocellulose membranes (0.2 µM, BioRad) at 100 V for 1 h. Membranes were blocked in 5% BSA or milk for 1 h RT depending on antibodies to be used. Membranes were incubated with primary antibodies overnight at 4° and washed for a total of 30 min in TBST before adding secondary diluted to 1:10,000 through 1:5,000 in 5% milk. Antibodies used in immunoblots are as follow: Anti-USP15 (Proteintech 14354-1-AP) used at 1:500-1:1000, Anti-Vinculin E1E9V XP (Cell Signaling Technologies #13901) used at 1:1000, Anti-KEAP1 P586 (Cell Signaling Technologies #4678) used at 1:500, Anti-NRF2 (Cell Signaling Technologies #12721) used at 1:500, anti-GAPDH D16H11 XP (Cell Signaling Technologies #5174) used at 1:1000, anti-pan-Actin (Cell Signaling Technologies #4968) used at 1:1000.
Precision plus dual color protein marker
Manufactured by Bio-Rad
Precision Plus Dual Color Protein Marker is a pre-stained protein standard used for estimating the molecular weight of proteins in SDS-PAGE analysis. It contains a mixture of 10 recombinant proteins with a molecular weight range of 10 to 250 kDa and is stained with two different dyes, allowing for the visualization of protein bands in two colors.
Automatically generated - may contain errors
Lab products found in correlation
Gradient gel, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Anti usp15, by Proteintech (1 mentions)
Anti vinculin e1e9v xp, by Cell Signaling Technology (1 mentions)
Anti gapdh d16h11 xp, by Cell Signaling Technology (1 mentions)
Anti pan actin, by Cell Signaling Technology (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Surepage bis tris gel, by GenScript (1 mentions)
Instantblue protein stain, by Abcam (1 mentions)
Pierce glycoprotein staining kit, by Thermo Fisher Scientific (1 mentions)
Odyssey clx system, by LI COR (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Amersham hybond ecl, by GE Healthcare (1 mentions)
Rabbit anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Fusion imaging system, by Vilber (1 mentions)
7 protocols using precision plus dual color protein marker
1
Western Blot Analysis of Cellular Signaling Proteins
2
Western Blot Immunodetection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A table of all antibodies is provided in the Key Resources Table . Antibody dilutions were prepared at 1:1000 to 1:500 in 5% BSA or dry milk in TBST. Protein samples prepared directly from lysate were prepped in 5X SDS sample buffer. Protein samples used in IPs were prepped in 2X Laemmli sample buffer reconstituted with BME. Immunoblotting was performed in SDS buffer, using BioRad gradient gels, at 175 V for 42 minutes. Precision plus dual color protein marker (BioRad) was used in parallel to samples. Proteins were wet-transferred onto nitrocellulose membranes (0.2 uM, BioRad) at 100V for 1 hr. Membranes were blocked in 5% BSA or milk for 1 hr RT depending on antibodies to be used. Membranes were incubated with primary antibodies overnight at 4 degrees and washed for a total of 30 minutes in TBST before adding secondary diluted to 1:10,000 in 5% milk.
3
Western Blot Immunodetection Protocol
4
Protein Characterization and Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified recombinant proteins from both expression systems were mixed with 4x NuPAGE™ sample buffer (Thermo Fisher). A final concentration of 10mM dithiothreitol was added to the sample buffer to reduce samples. Samples were heated for 10 minutes at 70°C and separated on a 4-20% SurePAGE™ Bis-Tris gel (GenScript) alongside a Precision Plus Dual Color protein marker (Bio-Rad). Total protein was visualized by staining the gel directly with InstantBlue™ protein stain (Abcam). Glycosylation of proteins was analyzed by staining the gel with the Pierce™ Glycoprotein Staining kit (Thermo Scientific). The amino acid sequences of the successfully produced proteins are presented in Supplementary Table 2
5
Plasmodium falciparum Gametocyte Antigen Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
P. falciparum NF54 gametocyte extract was prepared as described previously (34 (link)). The extract was mixed with 4x NuPAGE™ LDS sample buffer and heated for 10 minutes at 70°C before loading on a 4-12% Bis-Tris 2D-well gel. Precision Plus Dual Color protein marker (Bio-Rad) was used as size standard. Proteins were transferred to a 0.45 µm nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The blot was blocked with 5% skimmed milk in PBS and cut into strips. Each strip was incubated with a 1:100 dilution of pooled final bleed mice serum. After washing, the strips were incubated with 1:10,000 Goat Anti-Mouse IRDye 680RD (Li-cor, Cat. No. 926-68070). One strip was incubated with 1:100 polyclonal serum from rabbits immunized with R0. This strip was subsequently incubated with 1:10,000 Goat Anti-Rabbit IRDye 800CW (Li-cor, Cat. No. 926-32211). Strips were imaged using the Odyssey® CLx system (Li-cor).
+ Expand
P. falciparum NF54 gametocyte extract was prepared as described previously (34 (link)). The extract was mixed with 4x NuPAGE™ LDS sample buffer and heated for 10 minutes at 70°C before loading on a 4-12% Bis-Tris 2D-well gel. Precision Plus Dual Color protein marker (Bio-Rad) was used as size standard. Proteins were transferred to a 0.45 µm nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The blot was blocked with 5% skimmed milk in PBS and cut into strips. Each strip was incubated with a 1:100 dilution of pooled final bleed mice serum. After washing, the strips were incubated with 1:10,000 Goat Anti-Mouse IRDye 680RD (Li-cor, Cat. No. 926-68070). One strip was incubated with 1:100 polyclonal serum from rabbits immunized with R0. This strip was subsequently incubated with 1:10,000 Goat Anti-Rabbit IRDye 800CW (Li-cor, Cat. No. 926-32211). Strips were imaged using the Odyssey® CLx system (Li-cor).
6
SDS-PAGE Protein Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were prepared with 4X Laemmli sample buffer (BioRad Laboratories #161-0747) supplemented with 10% β-mercaptoethanol (Sigma-Aldrich #63689) and heated for 10 minutes (min) at 85°C before loading 15 μl/well on 12-well 4-20% TGX precast protein gels (Biorad Laboratories #4561095). As a marker 2 μl of the Precision Plus Protein dual color marker (BioRad Laboratories #1610374) was loaded, and the gel was run at 45V for 3 hours.
7
Western Blot Analysis of Na⁺V1.5 Channel Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells that heterologously expressed WT or mutant NaV1.5 channels were lysed and samples of equal total protein amount (standard BCA-assay) were run on 10% SDS-PAGE. The electrophoresed proteins were transferred to nitrocellulose-membrane (#RPN303D, Amersham Hybond ECL, GE Healthcare Europe). Subsequently, the membrane was cut between the 75 kDa and 50 kDa band (Precision Plus Protein dual color marker #161-0374, BIO-RAD). The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-NaV1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA). Goat anti-rabbit IgG HRP (#31463, Thermo Fisher, 1:20000 in 1xTBST with 1% BSA) or rabbit anti-mouse IgG HRP (#31455, Thermo Fisher; 1:25000 in 1x TBST with 1% BSA) were used as secondary antibody to visualize NaV1.5 and GAPDH by Super SignalTM West Pico/Femto Luminol (Thermo Fisher) and Fusion imaging system (Vilber Lourmat).
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!