Recombinant GST, GST-POZZNF509, GST-ZFZNF509L, GST-ZNF509L and GST-ZNF509S1 fusion proteins were prepared from Escherichia coli BL21 (DE3) cells by glutathione-agarose 4 bead affinity chromatography (Peptron). p300 polypeptide fragments were prepared in vitro using transcription and translation (TNT)-coupled wheat germ extracts in the presence of [35S]-methionine (Promega). GST-fusion protein-agarose bead complexes were then incubated with the labeled p300 polypeptide fragments at 4°C for 4 h in 25 mM HEPES, pH 7.6, 0.5 mM EDTA, 12.5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol and 0.2 mM phenylmethylsul fonyl fluoride (HEMG) buffer. Other GST protein pull-down procedures were performed as we have previously reported (15 (link)).
35s methionine
Manufactured by Promega
Sourced in United Kingdom
[35S]-methionine is a radioactively labeled amino acid used for in vitro protein synthesis and labeling experiments. It serves as a precursor for the incorporation of radioactive sulfur into newly synthesized proteins.
Automatically generated - may contain errors
Lab products found in correlation
Tnt quick coupled transcription translation system, by Promega (3 mentions)
Anti ha affinity gel, by Merck Group (2 mentions)
Tnt quick master mix, by PerkinElmer (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Image studio software, by LI COR (1 mentions)
35s methionine, by PerkinElmer (1 mentions)
Phosphocreatine, by Merck Group (1 mentions)
Creatine phosphokinase, by Merck Group (1 mentions)
Inorganic pyrophosphatase, by Merck Group (1 mentions)
8 protocols using 35s methionine
1
Characterization of ZNF509 and p300 Interaction
2
Purification and Pulldown of GST-Fusion Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid GEX-5X1 and pGEX-p50C, respectively, were transformed into E. coli BL21(DE) and overexpression of GST and GST-p50c fusion proteins was accomplished by induction with 400 μM isopropyl 1-thio-β-d-galactopyranoside (IPTG) for 3 h at 37°C. The proteins were purified using a GST purification module (Amersham Biosciences) according to the manufacturer's instructions.
Expression of 35S-labelled nsp12 in wheat germ extract by in vitro translation in the presence of [35S] methionine was carried out according to the recommended protocol (Promega). In the pulldown experiment, 30 μL of GST-Sepharose 4B or GST-p50C-Sepharose 4B Microbeads were added with 90 μL of lysis buffer (140 mM NaCl, 10 mM Tris-HCl (pH 8.0) and 0.5% Nonidet P-40)) for 1 h at room temperature. Sepharose 4B beads were washed 5 times with the lysis buffer and boiled in 2x SDS loading buffer for 7 min. The eluted pellets were then subjected to SDS-PAGE.
Expression of 35S-labelled nsp12 in wheat germ extract by in vitro translation in the presence of [35S] methionine was carried out according to the recommended protocol (Promega). In the pulldown experiment, 30 μL of GST-Sepharose 4B or GST-p50C-Sepharose 4B Microbeads were added with 90 μL of lysis buffer (140 mM NaCl, 10 mM Tris-HCl (pH 8.0) and 0.5% Nonidet P-40)) for 1 h at room temperature. Sepharose 4B beads were washed 5 times with the lysis buffer and boiled in 2x SDS loading buffer for 7 min. The eluted pellets were then subjected to SDS-PAGE.
3
Radiolabeled TNT Protein Synthesis
4
In Vitro Expression of PSA and PSMA Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 8
In vitro translation performed to confirm the expression of the PSA and PSMA antigens. The TNT® Quick Coupled Transcription/Translation System and 35S-methionine (Promega) were used. The pVAX vector alone (negative control) or pVAX backbone with the PSA or PSMA antigen inserts and 35S-methionine was added to the reaction mixture according to the manufacturer's instructions. The reaction was carried out at 30° C. for 2 bents. Labeled proteins were immunoprecipitated with anti-HA Affinity Gel (Sigma, St. Louis, Mo.) by rotation overnight in radioimmunoprecipitation assay (RIPA) buffer at 4° C. The immunoprecipitated proteins were electrophoresed on a SDS-PAGE gel that was subsequently fixed and dried. Expression of the 35S-labeled proteins was detected by autoradiography. The results are shown in
5
In vitro Validation of PSA and PSMA Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 8
In vitro translation performed to confirm the expression of the PSA and PSMA antigens. The TNT® Quick Coupled Transcription/Translation System and 35S-methionine (Promega) were used. The pVAX vector alone (negative control) or pVAX backbone with the PSA or PSMA antigen inserts and 35S-methionine was added to the reaction mixture according to the manufacturer's instructions. The reaction: was carried out at 30° C. for 2 hours. Labeled proteins were immunoprecipitated with anti-HA Affinity Gel (Sigma, St. Louis, Mo.) by rotation overnight in radioimmunoprecipitation assay (RIPA) buffer at 4° C. The immunoprecipitated proteins were electrophoresed on a SDS-PAGE gel that was subsequently fixed and dried. Expression of the 35S-labeled proteins was detected by autoradiography. The results are shown in
6
In Vitro Polyprotein Translation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polyprotein constructs were used with wheat germ transcription–translation system (TNT®, Promega) according to the manufacturer’s instructions in the presence of [35S]-Methionine (Amersham, Buckinghamshire, UK). Radiolabelled protein products were separated in 10% SDS–PAGE [57 (link)] and detected by autoradiography.
7
In vitro SUMO Modification Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro SUMO modification reactions were performed as indicated in Lee et al.49 . Recombinant SUMO and the other enzymes that will be used in the following experiments were previously expressed in E.Coli and purified as previously described49 . Briefly, cDNAs coding for SUMO-2 and GEMIN5 were transcribed and translated in rabbit reticulocyte lysate in the presence of [35S]methionine according to the manufacturer’s instructions (Promega). Assays were performed in the presence of high concentrations of E1 (220 nM) and E2 (600 nM) enzymes and contained 2 µl of translation product in a 10-µl reaction mixture containing 20 mM HEPES (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM dithiothreitol (DTT), 10 µM recombinant SUMO-1, 1 mM ATP, 5 mM phosphocreatine (Sigma), 20 U of creatine phosphokinase (Sigma)/ml, and 0.6 U/ml of inorganic pyrophosphatase (Sigma)/ml. Reactions were separated by SDS-PAGE and analyzed by autoradiography. The process was repeated with individual and combined mutants for lysines which are predicted target locations for sumoylation.
8
Precursor Import into Organelles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The precursors: pF1β (Nicotiana plumbaginifolia), pSSU (Spinacia oleracea) and AtThrRS(1–100)-RFP were expressed in a coupled transcription/translation system in the presence of [35S]-methionine according to the manufacturer’s instructions (Promega). AtThrRS-dTP(1–60) derived peptide fragments were synthesized by GenicBio (http://www.genicbio.com ). Mitochondria and chloroplasts were isolated as described in [40] and [41] , respectively. In vitro import reactions were performed according to [42] (link) and [41] . In order to study the effect of AtToc34 on in vitro import of AtThrRS(1–100)-RFP, purified AtToc34ΔTM252 and BSA (25, 50 and 75 μg) were added to the import reactions. In order to study the effect of AtThrRS-dTP(1–60) peptide fragments on the in vitro import of pF1β, and pSSU, isolated mitochondria and chloroplasts were pre-incubated with 20 μg of each peptide for 10 min on ice, prior to addition of the precursor. Import efficiency was calculated as a ratio between mature protein after PK treatment/input protein. To assess inhibition of import by peptide fragments, import efficiency of each reaction with peptide fragments was divided by the import efficiency of the reaction without peptides. Each experiment was repeated three times, gels were quantified and mean values were taken for calculation of inhibition of import.
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!