Protein samples were separated using NuPAGE Bis/Tris gels, as described [15 (link)], loading 2.5 × 106 parasite equivalents per lane. Membranes were probed with rat anti-HA antibodies (1:100 to 1:3,000 dilutions; clone 3F10, Sigma, 11867423001), rabbit anti-TgTom40 [54 (link)] (1:2,000 dilution), mouse anti-GFP (1:1,000 dilution; Sigma, 11814460001), mouse anti-BAG1 [55 (link)] (1:250 dilution; a kind gift from Louis Weiss, Albert Einstein College of Medicine), rabbit anti-SAG1 (1:1,000 dilution; a kind gift from Michael Panas and John Boothroyd, Stanford University), or mouse anti-TgGRA8 [56 (link)] (1:100,000 dilution; a kind gift from Gary Ward, U. Vermont) as primary antibodies, and horseradish peroxidase-conjugated goat anti-rat (1:5,000 to 1:10,000 dilutions; Santa Cruz, sc-2006, or Abcam, ab97057), goat anti-rabbit (1:5,000 to 1:10,000 dilution; Santa Cruz, sc-2004, or Abcam, ab97051), or goat anti-mouse (1:5,000 to 1:10,000 dilution; Santa Cruz, sc-2005) secondary antibodies.
Clone 3f10
Manufactured by Merck Group
The Clone 3F10 is a laboratory equipment product designed for cell-based assays. It is a high-affinity rat anti-HA (hemagglutinin) monoclonal antibody conjugated to horseradish peroxidase (HRP). The Clone 3F10 can be used to detect and quantify HA-tagged proteins in various experimental settings.
Automatically generated - may contain errors
3 protocols using clone 3f10
1
Western Blot Analysis of Parasite Proteins
2
Mammalianized Arginine Deiminase (ADI) Expression
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Example 1
ADI enzyme is widely expressed in bacteria and uses the substrates L-arginine and water to generate L-citrulline and ammonia. The sequence of bacterial ADI is provided in SEQ ID NO:1. The bacterial gene ADI (E.C. 3.5.3.6) was mammalianized through codon usage and fused to a hemagglutinin tag (HA tag). The sequence of mammalianized ADI is provided in SEQ ID NO: 2. A sequence alignment comparing the sequence of mammalianized ADI to that of the bacterial arginine deiminase is shown in
The mammalianized ADI and GFP were expressed in AAV9 using the vector pTR-ADIw, shown in
HeLa cells were transfected with AAV9-ADI or AAV9-EC to confirm mammalian expression of the viral construct. Following transient transfection, cells were harvested and protein expression was analyzed by Western blot using a rat anti-HA antibody (Clone 3F10 from Sigma Aldrich Cat#12158167001). As shown in
3
Parasite Protein Western Blotting
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Parasite protein preparations and associated western blotting was performed as described previously [59] (link). Membranes were probed with rat anti-HA (Sigma, clone 3F10; 1:500 dilution)
and rabbit anti-TgTom40 ([60]; 1:2,000 dilution) primary antibodies, and horseradish peroxidase-conjugated goat anti-rat (Abcam catalogue number ab97057; 1:5,000 dilution) and goat anti-rabbit (Abcam catalogue number ab97051; 1:10,000 dilution) secondary antibodies.
Blots were imaged on a ChemiDoc MP imaging system (Biorad).
and rabbit anti-TgTom40 ([60]; 1:2,000 dilution) primary antibodies, and horseradish peroxidase-conjugated goat anti-rat (Abcam catalogue number ab97057; 1:5,000 dilution) and goat anti-rabbit (Abcam catalogue number ab97051; 1:10,000 dilution) secondary antibodies.
Blots were imaged on a ChemiDoc MP imaging system (Biorad).
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