400 μg isolated mitochondria were resuspended in incubation buffer (1 mg/ml BSA, 20 μCi [α-33P] UTP (3000 Ci/mmol)). Subsequently, the samples were incubated for 1 h at 37°C on a rotating wheel. Mitochondria were re-isolated by centrifugation for 2 min at 9000 g, resuspended in 500 μL incubation buffer supplemented with 2 mM UTP and incubated for 10 min at 37°C. Samples were centrifuged at 9000 g for 4 min at 4°C and mitochondria washed twice with incubation buffer. Mitochondrial RNA was purified by the RNeasy Mini Kit (QIAGEN). Samples were mixed in a 1:2 ratio with RNA sample loading buffer (Sigma), incubated at 65°C for 15 min, followed by 2 min incubation on ice. Samples were analyzed using Formaldehyde-agarose gels (1.2% agarose (RNase free, Ambion), 2.2 M formaldehyde in NorthernMax MOPS gel running buffer (Ambion)). Gels were vacuum dried and analyzed by autoradiography. For staining of mitochondrial rRNAs, a wet transfer was performed in 20x SSC overnight onto a nylon membrane followed by staining with Methylene Blue (Molecular Research Center Inc.) for 10 min.
Northernmax mops gel running buffer
Manufactured by Thermo Fisher Scientific
NorthernMax MOPS gel running buffer is a pre-formulated solution designed for use in Northern blot analysis. It is used to create a stable, denaturing environment for the separation of RNA molecules by size during gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Rna sample loading buffer, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Prime it 2 random primer labeling kit, by Agilent Technologies (1 mentions)
Ssrna ladder, by New England Biolabs (1 mentions)
Northernmax denaturing gel buffer, by Thermo Fisher Scientific (1 mentions)
Amersham hybond n blot, by GE Healthcare (1 mentions)
Ultrahyb hybridization buffer, by Thermo Fisher Scientific (1 mentions)
2 protocols using northernmax mops gel running buffer
1
Mitochondrial RNA Labeling and Analysis
2
Northern Blot Analysis of FORCP mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2 μg polyA+ RNAs were isolated using NucleoTrap mRNA Mini kit for polyA+ RNA extraction (Macherey-Nagel) and separated by 1% formaldehyde agarose gel. ssRNA Ladder (New England Biolabs) was used for marker. The agarose gel was prepared using NorthernMax Denaturing Gel Buffer (Ambion) and run using NorthernMax MOPS Gel Running Buffer (Ambion). RNA gel was washed two times with nucleotide-free water for 30 min each, followed by transfer in 10x SSC buffer to Amersham Hybond-N+ blot (GE Healthcare). RNA was then fixed by UV crosslinking with 120 mJ/cm2. Labeling of random-primed probes was performed with the Prime-It II Random Primer Labeling Kit (Agilent) and a mammalian expression vector containing full-length FORCP cDNA. Hybridization was done overnight at 42°C in ULTRAhyb hybridization buffer (Ambion) as per the manufacture's instructions. Blots were washed at 42°C using 2X SSC+0.1% SDS and 0.1xSSC+0.1%SDS and imaged using a Phosphorimager.
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!