5 × 106P. falciparum NF54 sporozoites were resuspended in 100 μL 2× NuPAGE sample buffer supplemented with reducing agent (Thermo Fisher Scientific) and incubated at 95°C for 5 min, after which DNA was sheared. The resulting sporozoite protein extract was resolved on a 3%–8% Tris-Acetate SDS-polyacrylamide gel (NuPAGE) using MOPS SDS Running Buffer (Thermo Fisher Scientific) and transferred to a PVDF membrane. The membrane was blocked for 1 hr with 5% milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween 20). PfEMP1 proteins were detected using guinea pig anti-ATS antibodies (Nacer et al., 2015 (link)) or anti-NF54_SpzPfEMP1 antibodies. PfAldolase was detected with anti-PfAldolase-HRP (horseradish peroxidase) antibodies (Abcam, ab38905). Primary antibodies were detected by goat anti-guinea pig-HRP (Abcam, ab6908), anti-rat-HRP, or anti-mouse-HRP (GE Healthcare Life Sciences, NA935V and NA931V, respectively) secondary antibodies. HRP signal was developed with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, #34080) and imaged with a ChemiDoc XRS+ System (Bio-Rad). Images were analyzed using the Fiji image processing package (http://fiji.sc ).
Ab38905
Manufactured by Abcam
Ab38905 is a laboratory equipment product. It is a tool designed for use in scientific research and testing procedures. The core function of this product is to facilitate specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (2 mentions)
Ab6908, by Abcam (1 mentions)
Reducing agent, by Thermo Fisher Scientific (1 mentions)
Mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Na931v, by GE Healthcare (1 mentions)
Na935v, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Autoradiography film, by Avantor (1 mentions)
Goat anti rat hrp conjugate, by Merck Group (1 mentions)
Goat anti rabbit hrp conjugate, by Merck Group (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Mouse anti h3, by Abcam (1 mentions)
Ab9110, by Abcam (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using ab38905
1
Detecting Malaria Parasite Proteins
2
Western Blot Analysis of Subcellular Extracts
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Subcellular extract preparations were prepared as described previously50 (link). Equal amounts of protein were loaded and separated on 4–12% SDS-PAGE gel (Invitrogen), and analyzed by western blot using antibodies anti-PfSET7 (mouse), anti-HA (mouse, Roche 12CA5 or rat, Roche 3F10), anti-Pf-aldolase (Abcam ab38905) or anti-Histone H3 (mouse, Abcam ab1791), all at 1:2000 dilution. Secondary antibodies were anti-rabbit IgG-HRP (GE NA934V, Lot 9568295) and anti-mouse IgG-HRP (GE NA931V, Lot 9648752), both at 1:4000. Blots were developed using ECL (Thermo Scientific).
3
Parasite Protein Western Blot Assay
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Full parasite protein western blot samples were collected by lysing RBCs with 0.1% saponin and boiling protein in Loading Buffer (50mM Tris-Cl pH 8.0, 20% SDS, 1% Bromophenol Blue). Fractionated parasite protein was prepared as described in [95 (link)]. Blots were performed as described [19 (link)]. Primary antibodies used were: 1/1000 rat ant-HA (Roche 3F10), 1/1000 mouse anti-GFP (Roche), 1/3000 rabbit anti-aldolase conjugated to HRP (Abcam ab38905), or 1/3000 mouse anti-H3 (Abcam ab10799). Secondary antibody concentrations used were 1/3000 goat anti-rat HRP conjugate (Millipore), 1/3000 goat anti-mouse HRP conjugate, or 1/10,000 (Pierce) goat anti-rabbit HRP conjugate (Millipore). ECL reagent (Pierce) was used to detect HRP signal. Blots were exposed to autoradiography film (VWR) and visualized using an autoradiography developer.
4
Extraction and Analysis of Parasite Proteins
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Parasites were harvested using 0.15% saponin
lysis of infected RBC (iRBC), followed by PBS washes. Proteins of
the parasite pellets were extracted using lysis buffer (1 mM DTT,
2× Laemmli buffer (Bio-Rad, 161–0737), and protease inhibitor
cocktail (Roche, 11836170001) dissolved in PBS and sonicated for 5
min (30sec on/off cycles). The equivalent of 1 × 107 parasites was loaded per lane in a 4–20% Mini-PROTEAN TGX
Stain-Free gel (Bio-Rad, 4568096), and proteins were separated and
transferred onto a nitrocellulose membrane using the transblot semi-wet
transfer system (Bio-Rad). Blocking and antibody dilutions were performed
in PBS-Tween-20 with 5% skim milk. Anti-HA (Abcam, ab9110), HRP-anti-Plasmodium aldolase (Abcam, ab38905), and anti-H3
(Abcam, ab1791) were used at 1:1000; secondary anti-rabbit HRP antibody
was diluted at 1:5000. The blot was revealed using a Super Signal
West-Femto chemiluminescent substrate (Thermo Fisher Scientific).
lysis of infected RBC (iRBC), followed by PBS washes. Proteins of
the parasite pellets were extracted using lysis buffer (1 mM DTT,
2× Laemmli buffer (Bio-Rad, 161–0737), and protease inhibitor
cocktail (Roche, 11836170001) dissolved in PBS and sonicated for 5
min (30sec on/off cycles). The equivalent of 1 × 107 parasites was loaded per lane in a 4–20% Mini-PROTEAN TGX
Stain-Free gel (Bio-Rad, 4568096), and proteins were separated and
transferred onto a nitrocellulose membrane using the transblot semi-wet
transfer system (Bio-Rad). Blocking and antibody dilutions were performed
in PBS-Tween-20 with 5% skim milk. Anti-HA (Abcam, ab9110), HRP-anti-Plasmodium aldolase (Abcam, ab38905), and anti-H3
(Abcam, ab1791) were used at 1:1000; secondary anti-rabbit HRP antibody
was diluted at 1:5000. The blot was revealed using a Super Signal
West-Femto chemiluminescent substrate (Thermo Fisher Scientific).
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