Molecular cloning was performed using inFusion HD cloning technology (Clontech/Takara Biotech, Mountain View, CA). Constructs used for bacterial expression were sub-cloned into a modified pGEX vector to add GST-tags. For mammalian expression, constructs were inserted into pGLUE to add streptavidin binding protein/TEV/calmodulin binding protein tags; or pSNAPf to add SNAP-epitope tags; or pcDNA3.1 to fuse MYC tags. BG-782 SNAP substrate was from New England Biolabs (Ipswich, MA). PageRuler Prestained NIR Protein Ladder was used for all PAGE NIR (Thermo Fisher Scientific, Waltham, MA).
Pageruler prestained nir protein ladder
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The PageRuler Prestained NIR Protein Ladder is a protein ladder used for the estimation of molecular weights of proteins in near-infrared Western blotting applications. It provides a range of pre-stained protein bands that can be visualized using near-infrared detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Skim milk, by Merck Group (2 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (2 mentions)
Methanol, by Avantor (2 mentions)
Anti hisg hrp, by Thermo Fisher Scientific (2 mentions)
Epi red, by Syngene (2 mentions)
Run blot buffer, by Abcam (2 mentions)
Gradient polyacrylamide gels, by Abcam (2 mentions)
Instantblue, by Abcam (2 mentions)
Luminata forte, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Bg 782, by New England Biolabs (1 mentions)
Irdye 800cw donkey anti goat, by LI COR (1 mentions)
Irdye 680cw goat anti mouse, by LI COR (1 mentions)
Mini trans blot cell wet blotting system, by Bio-Rad (1 mentions)
Mini protean tetra cell electrophoresis chamber, by Bio-Rad (1 mentions)
Complete mini, by Roche (1 mentions)
6 protocols using pageruler prestained nir protein ladder
1
Molecular Cloning Using Fusion and Epitope Tags
2
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins (TP alone or TEV treated) were run in 12–4% SDS page in parallel. Half of the gel was analysed by Coomassie Blue staining, and the other half of the gel was moved onto a PVDF membrane and sandwiched inside two thick filter papers and run in Trans-Blot SD semidry electrophoresis cell (Bio-Rad, Glandesville, NSW, Australia) for 30 min at 12 V. The PVDF membrane was blocked by Odyssey® Blocking Buffer (Licor, Lincoln, NE, U.S.A.) and labelled with primary mouse Trx–tag Antibody (Assay Matrix PTY LTD) and secondary IRDye® 800 CW Goat anti-Mouse IgG (Licor, Lincoln, NE, U.S.A.) in TBST buffer (Tris-buffered-saline+ Tween 0.1% w/v) at the suggested dilutions by the manufactures. PageRuler™ Prestained NIR Protein Ladder (Thermofisher, Scoresby, VIC, Australia) was used as a molecular weight ladder.
3
Comprehensive Protein Visualization Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE was carried out using precast 4-20% gradient polyacrylamide gels (Expedeon) and stained with InstantBlue (Expedeon). The PageRuler Prestained NIR Protein Ladder (Thermo Scientific) was included. Proteins were transferred to PVDF membranes (Millipore) in run blot buffer (Expedeon) and 20% methanol (VWR), washed in TBS-T buffer and blocked in TBS-T with 5% skim milk (Sigma). Blots were incubated overnight at 4°C with agitation in blocking buffer and anti-HisG-HRP (1:5000, Invitrogen), or anti-V5-HRP (1:5000, Invitrogen). Chemiluminescent detection of blots was conducted with Luminata Forte (Millipore) in a G-box illuminator with Epi Red and FRLP filters (Syngene) for visualizing ladder and samples, respectively.
4
Molecular Cloning with InFusion HD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Molecular cloning was performed using inFusion HD cloning technology (Clontech/Takara Biotech, Mountain View, CA). The pSNAPf and pCLIP vector, as well as SNAP substrates, BG-782 and Alexa Fluor 488, and CLIP substrate, BC-680 were purchased from New England Biolabs (Ipswich, MA). PageRuler Prestained NIR Protein Ladder was used for all PAGE NIR analyses (Thermo Fisher Scientific, Waltham, MA).
5
Western Blot Analysis of p75 NGF Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was isolated using RIPA cell lysis buffer supplemented with protease inhibitors (Complete Mini, Roche, Basel, Switzerland). SDS-PAGE was performed using the Mini-PROTEAN® Tetra Cell electrophoresis chamber (BioRad, Hercules, CA, USA). The PageRuler Prestained NIR Protein Ladder (Thermo Fisher Scientific) was used. Separated proteins were blotted onto a nitrocellulose membrane (Amersham Protran 0.2 NC, Sigma-Aldrich) using the Mini Trans-Blot® Cell wet blotting system (BioRad). Membranes were incubated with primary antibodies (rabbit anti-rat p75 NGF receptor antibody; mouse anti-rat beta-actin antibody (both Abcam, Cambridge, UK)) overnight at 4 °C, and infrared-labeled secondary antibodies (IRDye 800CW donkey anti-goat, Li-Cor; IRDye 680CW goat anti-mouse (Li-Cor Biosciences, Lincoln, NE, USA)) for one hour at room temperature. Visualization was performed using the Odyssey® Infrared Imaging System.
6
Protein Detection and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE was carried out using precast 4-20% gradient polyacrylamide gels (Expedeon) and stained with InstantBlue (Expedeon). The PageRuler Prestained NIR Protein Ladder (Thermo Scientific) was included. Proteins were transferred to PVDF membranes (Millipore) in run blot buffer (Expedeon) and 20% methanol (VWR), washed in TBS-T buffer and blocked in TBS-T with 5% skim milk (Sigma). Blots were incubated overnight at 4C with agitation in blocking buffer and anti-HisG-HRP (1:5000, Invitrogen), or anti-V5-HRP (1:5000, Invitrogen). Chemiluminescent detection of blots was conducted with Luminata Forte (Millipore) in a Gbox illuminator with Epi Red and FRLP filters (Syngene) for visualizing ladder and samples, respectively.
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!