Purified MEK1 protein was separated by SDS–PAGE on a 4–15% gradient polyacrylamide gel (Bio-Rad) under reducing and denaturing conditions, and transferred to PVDF membranes. Membranes were blotted with either 1:2,500 Anti-His (6x-His Epitope TAG, PA1-983B, Thermo Fisher Scientific) for total MEK1 or 1:1,000 Anti-MEK1-SPSP (Phospho-MEK1 1/2 (Ser217/221), 9154, Cell Signaling Technology), followed by 1:10,000 DAR-HRP. Signal was detected by enhanced chemiluminescence (Bio-Rad) imaged on a ChemiDoc XRS+CCD camera. Densitometry was performed using the Bio-Rad Image Lab software.
Chemidoc xrs ccd camera
Manufactured by Bio-Rad
The ChemiDoc XRS+CCD camera is a high-performance imaging system designed for capturing and analyzing chemiluminescent and fluorescent samples in a laboratory setting. It features a charge-coupled device (CCD) sensor for sensitive image detection, providing researchers with accurate and reliable data for their experiments.
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6 protocols using chemidoc xrs ccd camera
1
Western Blot Analysis of Phosphorylated MEK1
2
Quantitative Western Blot Analysis
3
Western Blotting of Superfolder GFP
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For western analysis of sfGFP, 100 ng of total sfGFP samples were loaded on either 4–15% acrylamide gels (Bio-Rad) or on a handcast 10-well 12% acrylamide gel containing 25 μM Phos-tag acrylamide. Transferred PVDF membranes were blotted with 1:545 Anti-GFP (Invitrogen, Mouse Anti-GFP 33-2,600) followed by 1:10,000 DAM-HRP. Signal was detected by enhanced chemiluminescence (Bio-Rad) imaged on a ChemiDoc XRS+CCD camera. Densitometry was performed using the Bio-Rad Image Lab software.
4
Western Blot Analysis of Phosphorylated MEK1
5
Western Blot Analysis of sfGFP
6
Quantitative Western Blot Analysis
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Protein samples were subjected to electrophoresis on polyacrylamide gels and transferred to PVDF membranes. The membranes were incubated overnight in TBST blocking buffer containing 5% milk (w/v) (for the antibodies raised in sheep) or 3% BSA (w/v) (for all other antibodies). The membranes were immunoblotted with one of the following 1° antibodies in the appropriate blocking buffer for the denoted amount of time at RT: SPWNK1 S382 (2 hr.), TPNKCC1 (2 hr.), Strep-Tactin-HRP (1.5 hr.), or Anti-SPAK (1 hr). The membrane was then subjected to 3 washes in TBST for 5 min each, followed by the appropriate 2° antibody in blocking buffer for 1 hr at RT. The membranes were washed again 3 times in TBST for 5 min each. Signal was detected by enhanced chemiluminescence (Bio-Rad) imaged on a ChemiDoc XRS+ CCD camera.
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