6x-His Tag Monoclonal Antibody (4E3D10H2/E3) - 100 ug - MA1-135
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6x-His Tag Monoclonal Antibody (4E3D10H2/E3) - 100 ug - MA1-135
6x-His Tag Antibody (MA1-135) in ICC/IF
Immunofluorescent analysis of 6x-His Tag was performed using 70% confluent HEK-293 cells transfected with V5-H3-His construct. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with 6x-His Tag Monoclonal Antibody (4E3D10H2/E3) (Product # MA1-135) at 5 µg/mL and Histone H3 Recombinant Polyclonal Antibody (Product # 711055) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32733) respectively at a dilution of 1:2000 for 45 minutes at room temperature. Panel a (Nuclei: Red) represents 6x-His Tag Monoclonal Antibody (4E3D10H2/E3). Panel b (Nuclei: Green) represents Histone H3 Recombinant Polyclonal Antibody. Panel c (Nuclei: Blue) represents nuclei stained wirh ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel d represents the merged image showing the co-localization of nuclear signals in transfected cells. Panel e represents untransfected HEK cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Western Blot was performed using Anti-6x-His Tag Monoclonal Antibody (4E3D10H2/E3) (Product # MA1-135) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 60 µg (Lane 1), empty vector control, 60 µg (Lane 2), His-p65-YFP, 60 µg (Lane 3), His-p65-YFP, 40 µg (Lane 4), His-p65-YFP, 20 µg (Lane 5), HA-H3, 40 µg (Lane 6) and 25 ng Positope (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). A 92 kDa band of His-p65-YFP were observed in transfected lysates on probing with primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1500 (Product # A44241). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Positope (Product # R900-50) is a 53 kDa recombinant protein consisting multiple epitope tags, which has been used as a positive control for 6x-His Tag detection. No cross-reactivity was seen with HA-H3 expressing lysate. Western blot analysis of recombinant N-terminal His-tagged turbo GFP was performed by loading the indicated amounts of protein, and 15 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with StartingBlock (PBS) Blocking Buffer (Product # 37538) for at least 1 hour at room temperature. N-terminal His-tagged turbo GFP was detected at ~37 kD using a 6x-His Epitope Tag monoclonal antibody (Product # MA1-135) at a concentration of 2 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody (Product # 31439) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
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6x-His Tag Monoclonal Antibody (4E3D10H2/E3) - 100 ug - MA1-135
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