The coding region of mouse Mustn1 (NM_181390.3) and an mRNA sequence containing 5’ and 3’ untranslated regions (AK003945.1) were amplified from muscle RNA using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The resulting PCR products were ligated into intermediate vector pJET1.2/blunt (Thermo Fisher Scientific). The CDS was then cloned into expression vector pFLAG-CMV-5a (Sigma–Aldrich; contains a C-terminal FLAG-tag) and together with a N-terminal FLAG-tag into expression vector pcDNA3.1 (Invitrogen). The mRNA sequence (untagged) was cloned into expression vector pCMV5 (Dr. David Russell, UTSW). The sequences of all constructs were verified.
Pflag cmv 5a
Manufactured by Merck Group
Sourced in United States
PFLAG-CMV-5a is a lab equipment product manufactured by Merck Group. It is a device used in laboratory settings for a specific function. However, a detailed description of its core function cannot be provided in an unbiased and factual manner without the risk of extrapolation or interpretation. Therefore, the description for this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Quikchange site directed mutagenesis method, by Agilent Technologies (2 mentions)
Pjet1.2 blunt, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Pdsred er, by Takara Bio (1 mentions)
Pdsred monomer golgi, by Takara Bio (1 mentions)
Flag antibody, by Cell Signaling Technology (1 mentions)
Mab1501, by Merck Group (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using pflag cmv 5a
1
Mouse Mustn1 Cloning and Expression
2
GLT8D1 Expression Construct Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A full-length coding region of GLT8D1 was cloned into pFLAG-CMV-5a (Sigma-Aldrich, St. Louis, MO) to generate the wild-type (WT) GLT8D1 expression construct. The 2 GLT8D1 variations, p.I290M (c.870C>G) and p.R92C (c.274 G>A), were separately introduced into the WT expression plasmids by using the QuikChange Site-Directed Mutagenesis method (Agilent, Santa Clara, CA). The endoplasmic reticulum (ER) marker pDsRed-ER and the Golgi marker pDsRed-Monomer-Golgi were purchased from Clontech (Mountain View, CA). HEK293T cells were maintained in Dulbecco modified eagle medium supplemented with 10% FBS. Transient transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific).
3
Cloning and Validation of ORFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The open reading frame (ORF) encoding full-length DJ-1 was amplified from HEK293 cells cDNA, while the ORFs encoding the N-terminal domain of LRRK2 (amino acids 1–266, [25 (link)]) and full-length α-synuclein were amplified from pre-existing plasmids (Addgene: pDEST53-LRRK2-WT #25044 and EGFP-alpha synuclein-WT #40822, respectively). All ORFs were inserted into pFLAG-CMV-5a (Sigma-Aldrich) and pcDNA3.1(+)-PARP1cd [23 (link)] vectors. ORFs encoding α-synuclein mutants (E46K and A53T) were generated by PCR-based site-directed mutagenesis. All cloned DNA sequences were verified by DNA sequence analysis.
4
NIPA1 Mutant Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A human NIPA1 cDNA clone was purchased from TransOMIC (BC156247; Huntsville, AL, USA). The full‐length coding region of NIPA1 was cloned into pFLAG‐CMV‐5a (Sigma‐Aldrich, St. Louis, MO, USA) to generate the wild‐type NIPA1 expression plasmids. The NIPA1 mutations, c.316G>A and c.316G>C were introduced into the wild‐type expression plasmids, separately, by using QuikChange Site‐Directed Mutagenesis method (Stratagene; Agilent, Santa Clara, CA, USA). The plasmids expressing wild‐type, each one of the two mutant NIPA1, or empty vector were transfected into HEK293T cells, respectively, using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Forty‐eight hours post‐transfection, cells were harvested and analyzed by western blotting. The steady‐state NIPA1 expression levels were analyzed using the FLAG antibody (#8146, Cell Signaling Technology, Danvers, MA, USA). Actin was used as a loading control to ensure an equal amount of protein loading (MAB1501; Merck Millipore, Burlington, MA, USA).
5
Characterizing CSF1R Variants in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A human CSF1R cDNA clone was purchased from TransOMIC (BC047521; Huntsville, AL). The full‐length coding region of human CSF1R was cloned into pFLAG‐CMV‐5a (Sigma‐Aldrich, St. Louis, MO) to generate the wild‐type CSF1R expression plasmids. Nine CSF1R variants were introduced into the wild‐type expression plasmids, separately, using the QuikChange Site‐Directed Mutagenesis method (Stratagene; Agilent, Santa Clara, CA). These variants were p.T79M (c.236C > T), p.E478K (c.1432G > A), p.T507_H508insP (c.1520_1522dupCGC), p.K586* (c.1754dupT), p.G589R (c.1765G > A), p.R777Q (c.2330G > A), p.R782C (c.2344C > T), p.M875T (c.2624T > C), and p.A914T (c.2740G > A). CSF1R p.M875T was a well‐known pathogenic mutation5 and served as a positive control in this study; the remaining eight variants were identified in the present study (Table 1 ). HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a humidified incubator at 37°C under 5% CO2.
6
Cloning of LOX and LOX-v2 Expression Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression plasmids of LOX and LOX-v2 were constructed as previously described [6] . Briefly, the cDNAs encoding the processed LOX (codons 169-417) and the full-length LOX-v2 were PCR-amplified from the cDNA of HEK293 cells using Pfu polymerase (Stratagene, La Jolla, CA, USA) according to the manufacturer's suggestions. The PCR conditions consisted of 30 cycles at 94℃ for 60 s, 56℃ for 60 s, and 72℃ for 60 s, with a predenaturation at 94℃ for 4 min and a final extension at 72℃ for 7 min. The PCR amplified DNA fragments were gel-purified and then subcloned into pET21a (Novagen, Madison, WI, USA) and pFLAG-CMV-5a (Sigma-Aldrich, St. Louis, MO, USA). The oligonucleotide primers used for constructions of pET21a-LOX, pET21a-LOX-v2, pFLAG-LOX, and pFLAG-LOX-v2 are listed in Table 1. Unique restriction sites, NheI or HindIII for pET21a and EcoRI, BamHI, or KpnI for pFLAG-CMV-5a, were introduced in the primers for convenient subcloning (Table 1). Sequence fidelity of all the constructs was confirmed by DNA-sequencing analysis, using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer's protocol.
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!