RIPA lysis buffer (Sigma, St Louis, MO) composed of protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA) was used for protein lysis. The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to calibrate protein concentrations. SDS-NuPAGE treatment of 10–20 μg of protein were ran on 4–12% Bis-Tris Gel (Novex by Life Technologies, Carlsbad, CA) and transferred to PVDF membranes. Primary incubation with anti-Shpk antibody (Sigma; 1:2000) ran at 4 °C overnight. The next day, membranes were washed three times in Protein Wash Buffer (Azure Biosystems, Dublin, CA) and incubated in HRP-conjugated secondary antibodies goat anti-rabbit and rhodamine anti-tubulin (BioRad; 1:2000) for 1 h at RT. Membranes are again washed three times and a final time in 1× tris buffered saline (TBS). Blots were activated by HRP substrate Radiance Plus ECL Reagent (Azure) and chemiluminescent detection was captured on the c600 Imager (Azure). Western blots were quantitated by comparative densitometry against tubulin loading control.
Radiance plus ecl reagent
Manufactured by Azure Biosystems
Radiance Plus ECL Reagent is a chemiluminescent substrate designed for the detection of horseradish peroxidase (HRP) in Western blotting applications. The reagent generates a luminescent signal proportional to the amount of target protein, allowing for sensitive and quantitative protein detection.
Automatically generated - may contain errors
Lab products found in correlation
C600 imager, by Azure Biosystems (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protein wash buffer, by Azure Biosystems (3 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti shpk antibody, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sc 20150, by Abcam (1 mentions)
Ab3523, by Abcam (1 mentions)
Ab53444, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
3 protocols using radiance plus ecl reagent
1
Western Blot Analysis of Shpk Protein
2
M1 and M2 Macrophage Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was harvested and resuspended in 1% SDS from cultures of 1 × 106 BMDMs and IC-21 using TRIzol Reagent (Invitrogen) per manufacturers protocol and quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). 10–20 μg of protein was subjected to SDS-PAGE, transferred to a PVDF membrane, blocked in 5% Protein Blocking Reagent (Azure) for 1 hr at RT and then probed overnight with primary antibodies at 4 °C. Antibodies included M(LPS/IFNγ) markers iNOS (abcam ab3523; 1:1,000) and MCP1(CST 2029S; 1:500), M(IL-4/IL-10) markers ARG1 (Santa Cruz sc-20150; 1:200), CD206 (abcam ab64693; 1:500), macrophage marker CD68 (abcam ab53444; 1:500) and loading control Gapdh (abcam ab8245; 1:1,000). The following day, blots were washed three times in Protein Wash Buffer (Azure) and probed with HRP-conjugated secondary antibody of the appropriate species at 1:2,500 for one hour at room temperature. Blots were then washed three times in Protein Wash Buffer and once with TBS, developed with Radiance Plus ECL Reagent (Azure), and imaged on the c600 Imager (Azure).
3
Western Blot Analysis of Shpk Protein
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!