Mouse fusion constructs were generated using enhanced green fluorescent protein (GFP), monomeric red fluorescent protein (RFP) or monomeric cherry (Ch). The expression constructs for RFP-DNMT1, GFP-DNMT1 wt, GFP-DNMT1Δ356-404, GFP-DNMT1Δ458-500, GFP-NTD, Ch-TS, GFP-DNMT1 (1-308), GFP-TS, GFP-DNMT1 (629-1110) and GFP-CTD have been described previously (9 (link),21 (link),26 (link),40 (link),60 (link),61 (link)). GFP-DNMT1 deletion and HSANIE point mutants as well as UHRF1-GFP deletion expression constructs were derived from the corresponding wt constructs by overlap extension PCR (62 (link)). The GFP-UHRF1, UHRF1-GFP, Ch-UHRF1 and GFP-UHRF1 single domain constructs have been reported before (17 (link),51 (link),63 (link),64 (link)). GFP, RFP and RFP-PCNA have been reported before (9 (link),25 ,65 (link),66 (link)). All constructs were verified by DNA sequencing. The following monoclonal antibodies were used for immunoblotting: rat anti-RFP/Ch (5F8, Chromotek; (67 (link)), rat anti-GFP (3H9, Chromotek). In dependence on the expected intensity of the signals, secondary antibodies either conjugated to horseradish peroxidase (HRP) (anti-rat (Dianova)) or conjugated to fluorescent dyes (anti-rat Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 647 (Invitrogen)) were applied. For detection of HRP-conjugated antibodies an ECL Plus reagent (GE Healthcare, Thermo Scientific) was used.
Rat anti gfp 3h9
Manufactured by Proteintech
Sourced in United States
Rat anti-GFP (3H9) is a laboratory reagent designed to detect and bind to green fluorescent protein (GFP) in various biological samples. It is a monoclonal antibody produced in rats.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Gfp trap coupled agarose beads, by Proteintech (1 mentions)
Rabbit anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti myc tag 9b11, by Cell Signaling Technology (1 mentions)
Rabbit anti β actin, by Novus Biologicals (1 mentions)
Pdvf membrane, by Bio-Rad (1 mentions)
Mouse anti v5, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha, by Fortrea (1 mentions)
4 protocols using rat anti gfp 3h9
1
Fluorescent Protein-Tagged Construct Generation
2
GFP-Trap Protein Pulldown Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 100 OD600 of log-phase cells grown exponentially in YPD were harvested and washed twice with water then lysed by beating with 0.5-mm-diameter glass beads (Sigma-Aldrich) and ice cold, freshly prepared native lysis buffer (20 mM HEPES, pH 7.5, 0.5% Triton X-100, 200 mM NaCl, 1X protease inhibitor mix, 1 mM 1,10 phenanthroline, 1 mM EDTA, 10 mM iodoacetamide). To pulldown GFP-tagged proteins, lysates were incubated for 2 hours at 4°C with 25 μL GFP-Trap coupled agarose beads (Chromotek). After incubation, the agarose beads were washed three times in lysis buffer before samples were eluted with 2X SDS sample buffer. Nitrocellulose membranes were probed with rat anti-GFP (3H9, Chromotek, 1:2,500), PAP (Sigma-Aldrich, 1: 2,500), and mouse anti-HA (Y-11, Santa Cruz Biotechnology, 1:2,500) primary antibodies.
3
Immunoblotting Antibody Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [26 (link)] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [19 (link)] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology). Probes used for immunofluorescence: Alexa 633-conjugated concanavalin A (ConA) (Thermo Scientific). Antibodies used for flow cytometry: mouse anti-MHC I W6/32 (Novus Biologicals); mouse anti-NGFR (Sigma-Aldrich); and Alexa 633-conjugated goat anti-mouse IgG (Thermo Scientific).
4
Quantitative Protein Analysis via Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein lysates were prepared in lysis buffer containing protease inhibitors. Proteins were size-separated by SDS-PAGE and transferred onto nitrocellulose or PDVF membranes (Biorad). For Western blot analysis, primary antibodies (Supplementary Table 2 ) were rat anti-GFP (3H9) (1:1000, Chromotek), mouse anti-V5 (1:5000, Invitrogen), mouse anti-HA (1:5000, Covance) and mouse anti-α-tubulin (AA4.3) (1:1000, Developmental Studies Hybridoma Bank).
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!