Samples destined for Native-PAGE analysis were combined with native sample buffer (50 mM Tris-HCl, pH 7.4, 10% glycerol, 0.0014% xylene cyanol at final concentration) and loaded onto polyacrylamide gels [90 mM Tris-borate, pH 8.35, 5 mM MgCl2, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 2.5% sucrose] with 3% stacking and 4% resolving portions. Electrophoresis was performed with native running buffer (90 mM Tris-borate, pH 8.35, 5 mM MgCl2, 0.5 mM EDTA) at 100 V for 3 h on ice. Samples prepared with 20 mM ATP were separated using gels and running buffer containing 1 mM ATP. Samples intended for SDS-PAGE analysis were combined with Laemmli sample buffer [39 (link)] and resolved on 12% gels. Where necessary, gels were stained with Coomassie Brilliant Blue R-250 or silver nitrate [13 (link)]. Alternatively, proteins resolved by Native-PAGE or SDS-PAGE were transferred onto nitrocellulose membrane (PALL BioTrace™; 66,485) and immunoblotted with specific antibodies. Antibodies used in this study include anti-FLAG (Sigma; F1804), anti-PBA1 (Abcam; ab98861), anti-RPN1 (Abcam; ab98865), anti-HSP70 (Agrisera; AS08 371), anti-PA200 (Agrisera; AS19 4269), goat anti-mouse conjugated to horseradish peroxidase (HRP) (Cell Signaling; 7076S), and goat anti-rabbit conjugated to HRP (Sigma; A9169). Chemiluminescent signals were quantified using ImageJ (https://imagej.nih.gov/ij ).
Ab98861
Manufactured by Abcam
Sourced in Sweden
Ab98861 is a lab equipment product. It is a device used for measuring and analyzing samples in a laboratory setting. The core function of this product is to perform specific measurements or analyses as required by the user.
Automatically generated - may contain errors
4 protocols using ab98861
1
Native and SDS-PAGE Protein Analysis
2
Separation and Detection of ATG8, NBR1, RBCL, and PBA1
3
Separation and Detection of ATG8, NBR1, RBCL, and PBA1
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For SDS-PAGE and immunoblotting, seedlings were weighed, frozen, and ground in liquid nitrogen and incubated with the lysis buffer containing 0.125 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol for 2 hours at 65°C. Samples were normalized by adjusting the lysis buffer volumes based on the fresh tissue weight. After incubation, total cell extracts were centrifuged at 14,000 rpm for 10 min at room temperature to remove the tissue debris.
To compare the total cellular protein levels in different samples and to separate ATG8 and ATG8-PE, a Urea-Tricine-SDS-PAGE system was utilized59 (link). Proteins were then transferred onto a PVDF membrane and probed with a polyclonal ATG8 antibody prepared in house. ATG8 antibody was prepared as described60 (link). To detect NBR1, RBCL, and PBA1, protein samples were separated by standard SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with specific antibodies (anti-NBR1, AS142805, Agrisera; anti-RBCL AS03037, Agrisera; anti-PBA1, ab98861, Abcam).
To compare the total cellular protein levels in different samples and to separate ATG8 and ATG8-PE, a Urea-Tricine-SDS-PAGE system was utilized59 (link). Proteins were then transferred onto a PVDF membrane and probed with a polyclonal ATG8 antibody prepared in house. ATG8 antibody was prepared as described60 (link). To detect NBR1, RBCL, and PBA1, protein samples were separated by standard SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with specific antibodies (anti-NBR1, AS142805, Agrisera; anti-RBCL AS03037, Agrisera; anti-PBA1, ab98861, Abcam).
4
Quantitative Immunodetection of Photosynthetic Proteins
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Protein samples were extracted from seedlings at 0 HAL, 1 HAL, 2 HAL, 4 HAL, 8 HAL and 12 HAL after being pulverized in liquid nitrogen. SDS-PAGE and immunoblot were performed as described by Liang et al. (2018) and Lee et al. (Lee et al., 2013) . The experiment was repeated for three times with total protein extracts from three independent sets of cotyledon samples. For immunogold labeling, thin sections (80 nm thick) of HM20 embedded samples at each time point were prepared by ultramicrotomy, the following immunodetection of gold particles were performed according to the protocol explained in Wang et al. (Wang et al., 2017) . Antibodies for PsaD (AS09 461), Lhca (AS01 005), AtpB (AS05 085), AtpC (AS08 312), PetC (AS08 330), PORA (AS05 067), CURT1A (AS08 316), CURT1B (AS19 4289), and CURT1C (AS19 4287) were purchased from Agrisera (Agrisera, Sweden).
Anti-PBA1 antibody (ab98861) and anti-GFP antibody (ab6556) were purchased from Abcam (Abcam, USA). Antibodies against PsbO was provided by Michael Seibert (National Renewable Energy
Anti-PBA1 antibody (ab98861) and anti-GFP antibody (ab6556) were purchased from Abcam (Abcam, USA). Antibodies against PsbO was provided by Michael Seibert (National Renewable Energy
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