We used the same method as before to perform Western blotting.37 Before Western blotting, we used a total protein extraction kit (BC3710, Solarbio), cellular mitochondrial isolation kit (C3601, Beyotime) or tissue mitochondrial isolation kit (C3606, Beyotime), and nuclear protein extraction kit (R0050, Solarbio) to extract total protein, mitochondrial protein or de‐mitochondrial cytoplasmic protein, and nuclear protein, respectively. The primary antibodies used were anti‐PARP‐1 (1:1000, DF7198, Affinity); anti‐AIF (1:1000, BF0591, Affinity); anti‐COX IV (1:1000, AF5468, Affinity); anti‐Histone H3 (1:1000, AF0863, Affinity); anti‐β‐actin (1:10,000, 66009‐1‐Ig, Protentech); anti‐LC3BI/II (1:1000, A5402, Affinity); anti‐PINK1 (1:200, PA5‐85930, Invitrogen); anti‐Parkin (1:200, 702785, Invitrogen); anti‐SIRT3 (1:1000, Affinity, AF5135); anti‐SOD2/MnSOD (acetyl K68) (1:5000, ab137037, Abcam); and anti‐SOD2/MnSOD (1:5000, ab13533, Abcam). The second antibodies used were HRP‐conjugated AffiniPure Goat Anti‐Mouse IgG (1:10,000, Proteintech) and HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG (1:10,000, Proteintech). Finally, PVDF molds were visualized on a Tanon 2500R gel imaging system (Tanon) using a developer (Tanon), and the band intensity was quantified using ImageJ 1.39V software.
Af5135
Manufactured by Affinity Biosciences
Sourced in China
The AF5135 is a laboratory instrument designed for DNA and RNA extraction, purification, and quantification. It utilizes a magnetic bead-based technology to efficiently isolate nucleic acids from a variety of sample types. The core function of the AF5135 is to provide a reliable and automated solution for extracting high-quality nucleic acids for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Df7198, by Affinity Biosciences (2 mentions)
Bf0591, by Affinity Biosciences (2 mentions)
Ab13533, by Abcam (1 mentions)
Ab137037, by Abcam (1 mentions)
Affinipure goat anti mouse igg, by Proteintech (1 mentions)
Af0863, by Affinity Biosciences (1 mentions)
C3601, by Beyotime (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
C3606, by Beyotime (1 mentions)
Bc3710, by Solarbio (1 mentions)
R0050, by Solarbio (1 mentions)
Ab104224, by Abcam (1 mentions)
Df6278, by Affinity Biosciences (1 mentions)
G1128, by Wuhan Servicebio Technology (1 mentions)
Df13268, by Affinity Biosciences (1 mentions)
Af0217, by Affinity Biosciences (1 mentions)
Hrp conjugated goat anti rabbit igg h l, by ZenBio (1 mentions)
G1204, by Wuhan Servicebio Technology (1 mentions)
Df6701, by Affinity Biosciences (1 mentions)
Ab131368, by Abcam (1 mentions)
5 protocols using af5135
1
Protein Extraction and Western Blotting for Subcellular Analysis
2
Immunostaining of SIRT3, PARP-1, and AIF in Neurons
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VSC4.1 neurons or spinal cord frozen sections were fixed using 4% paraformaldehyde, then incubated with 0.3% Triton X‐100 for 10 min, bovine serum albumin (BSA) for 2 h, then incubated with primary antibody overnight at 4°C and with secondary antibody at room temperature for 2 h. In the end, the sections were blocked by incubating with DAPI at room temperature for 10 min. The primary antibodies are as follows: anti‐SIRT3 (1:500, AF5135, Affinity); anti‐PARP‐1 (1:500, DF7198, Affinity); anti‐AIF (1:500, BF0591, Affinity); anti‐NeuN (1:1000, ab104224, Abcam); anti‐β‐tubulin (1:500, 66240‐1‐Ig, Proteintech) The second antibodies are as follows: Alexa Fluor 488/568 goat anti‐mouse IgG, Alexa Fluor 568 goat anti‐rabbit IgG (1:1000, Thermo Fisher Scientific).
3
Immunohistochemical analysis of protein expression
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Briefly, the human NP tissues embedded in paraffin were cut in 15 μm sections. Then, the sections were deparaffinized with environment-friendly de-paraffin liquid (G1128, Servicebio, China) and dehydrated using gradient alcohol. The membrane-breaking solution (G1204, Servicebio, China) was used under the protocols. The sections were subsequently incubated with 3% BSA for 25 min to block the endogenous peroxidase, then with primary antibody against Sirt3 (#AF5135, Affinity, China, 1:200), GPX4 (#DF6701, Affinity, China, 1:200), FTH (#DF6278, Affinity, China, 1:50), ADAMTS5 (#DF13268, Affinity, China, 1:150), and MMP3 (#AF0217, Affinity, China, 1:100), at 4 °C overnight. On the second day, the sections were incubated with HRP-conjugated Goat Anti-Rabbit IgG H&L (511,203, ZENBIO, China, 1:300) for 1 h, finally the counterstaining was performed with hematoxylin solution for 5 min. The images of stained sections were obtained using the light microscopy (BX43, Olympus, Japan).
4
Protein Extraction and Western Blot Analysis
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The total protein was abstracted by adding the corresponding pyrolysis solution (4°C for 30 minutes) (28–9425–44, ReadyPrep; GE Healthcare Life Sciences). After centrifugation at 876×g for 10 minutes, the supernatant was collected. Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were processed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later transferred onto nitrocellulose membrane as previously described [11 (link)]. After blocking with 5% nonfat milk (with Tween-20) at room temperature for 2 hours, the membrane were incubated with the primary antibodies against GAPDH (1:1000, TA-08, ZSbio, China), VEGF (1:1000, bs-1313R, Bioss), Sirt3 (1:1000, AF5135, Affinity), and LC3 (1:1000, ab48394, Abcam) overnight at 4°C. The secondary antibody (1:10 000, ab131368, Abcam, USA) was incubated with the membrane for 2 hours at room temperature. The expression of target proteins was normalized to GAPDH. At least 6 repeats were included in the western blotting.
5
Immunohistochemical Analysis of Xenograft Tumor Samples
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Immunohistochemical reactions were carried out using the labeled streptavidin-biotin method. Tumors dissected from mouse xenograft models were fixed in 10% formalin solution overnight at 4 °C, and sections of 6 μm were cut and embedded in paraffin. Deparaffinized sections were heated for 5 min at 100 °C in a pressure cooker to reactivate the antigens and blocked with hydrogen peroxide. After being washed three times, the samples were incubated with antibodies at 4 °C overnight. The primary antibodies included anti-ACSS1 (Affinity, DF3727, 1:200), anti-VDAC1 (Affinity, DF6140, 1:200), and anti-SIRT3 (Affinity, AF5135, 1:200). After incubation with a secondary antibody at room temperature for 1 h, the samples were washed, and staining was developed by incubation with a DAB substrate kit (Pierce). Immunostained sections were assessed and photographed under a microscope at magnification of 200× (IX71, Olympus, Japan).
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