Cells were lysed in RIPA buffer. Total protein concentration was determined by DC™ Protein Assay kit (Bio-Rad). A total of 30 µg of protein was separated using 4-15% gradient SDS-PAGE (4-15% Ready Gel® Tris-HCL Gel; Bio-Rad) and transferred onto a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was probed with primary antibodies that specifically recognized HA (mouse anti-HA.11 clone 16B12 monoclonal 1:1,000; Covance) and α-tubulin (mouse anti-α-tubulin clone DM1A monoclonal; Sigma). The membrane was then incubated with secondary antibody (HRP-conjugated goat anti-mouse 1:10,000; Abcam) after thorough washing and visualized with a Western Lighting™ Ultra Chemiluminescence Substrate kit (PerkinElmer) using the ChemiDoc™ XRS+ system (Bio-Rad). The quantification of MagA-HA was performed using ImageLab™ software (Bio-Rad) and was normalized to α-tubulin.
Western lighting ultra chemiluminescence substrate kit
Manufactured by PerkinElmer
The Western Lighting™ Ultra Chemiluminescence Substrate kit is a laboratory product designed for the detection and visualization of proteins in Western blot analysis. It contains a chemiluminescent substrate that enables the sensitive and accurate quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ready gel tris hcl gel, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse, by Abcam (1 mentions)
Mouse anti ha 11 clone 16b12 monoclonal, by Fortrea (1 mentions)
Membrane filter, by Merck Group (1 mentions)
Protran, by Cytiva (1 mentions)
2 protocols using western lighting ultra chemiluminescence substrate kit
1
Quantitative Western Blot Analysis of MagA-HA
2
Plasmid Delivery and Protein Expression in Pseudomonas
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Plasmid pBAM1
and its derivatives were delivered from E.
coli CC118λpir (pBAM1 or pBAM1-RparsM-gfp) donor cells into P. putida KT2440 by tripartite
mating with the assistance of the helper strain E. coli HB101 (pRK600).21 (link) The conjugation mixture
was incubated at 30 °C for 6 to 8 h by tripartite mating on membrane
filters (0.45 μm, Millipore) on LB agar plates. The filter was
transferred to 5 mL of 10 mM MgSO4 and vortexed to suspend
the cells. Afterward, appropriate dilutions were plated onto selective
medium as indicated for counter-selecting against the donor cells.
The conjugation mixture was plated on minimal selective medium as
indicated. P. putida KT2440 containing different
expression vectors were cultured overnight in LB medium at 30 °C.
Cells were harvested and identified by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE). Western blot analysis was used to
probe the expression of RparsM. Proteins separated
by SDS PAGE were transferred to a nitrocellulose membrane (Protran,
Schleicher & Schuell). Western blot analysis was performed using
a Western Lighting Ultra chemiluminescence substrate kit (PerkinElmer)
using an antimouse IgG to the six histidine tag.
and its derivatives were delivered from E.
coli CC118λpir (pBAM1 or pBAM1-RparsM-gfp) donor cells into P. putida KT2440 by tripartite
mating with the assistance of the helper strain E. coli HB101 (pRK600).21 (link) The conjugation mixture
was incubated at 30 °C for 6 to 8 h by tripartite mating on membrane
filters (0.45 μm, Millipore) on LB agar plates. The filter was
transferred to 5 mL of 10 mM MgSO4 and vortexed to suspend
the cells. Afterward, appropriate dilutions were plated onto selective
medium as indicated for counter-selecting against the donor cells.
The conjugation mixture was plated on minimal selective medium as
indicated. P. putida KT2440 containing different
expression vectors were cultured overnight in LB medium at 30 °C.
Cells were harvested and identified by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE). Western blot analysis was used to
probe the expression of RparsM. Proteins separated
by SDS PAGE were transferred to a nitrocellulose membrane (Protran,
Schleicher & Schuell). Western blot analysis was performed using
a Western Lighting Ultra chemiluminescence substrate kit (PerkinElmer)
using an antimouse IgG to the six histidine tag.
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