SW620/HCT116 were lysed for 30 mins in ice-cold RIPA lysis buffer for protein extraction according to the manufacturer’s protocol. Equal amounts of protein per sample (10 µg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes. After blocking with 5% non-fat milk for 1 hr at room temperature, the membranes were incubated overnight with polyclonal rabbit anti-human ENO1(A1033; 1:1000 dilution; ABclonal; Wuhan, China) or Rab1A (1:2000, Abcam, USA, Ab97956) and mouse anti-human GAPDH antibodies, followed by HRP-conjugated secondary antibodies for 1 hr at room temperature. The immunoreactive bands were visualized by chemiluminescence and quantified using ImageJ software.
Ab97956
Manufactured by Abcam
Sourced in United States, China
Ab97956 is a laboratory product manufactured by Abcam. It is a piece of lab equipment. No further details on its core function or intended use are available.
Automatically generated - may contain errors
Lab products found in correlation
Tissue staining kit, by Zhongshan Biotechnology (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Anti gm130, by BD (1 mentions)
Ab9106, by Abcam (1 mentions)
Rabbit anti mouse igg, by Agilent Technologies (1 mentions)
Sab4200135, by Merck Group (1 mentions)
Alexa fluorophore, by Thermo Fisher Scientific (1 mentions)
Ab108338, by Abcam (1 mentions)
Ab155686, by Abcam (1 mentions)
Pab160011, by Bioswamp (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
5 protocols using ab97956
1
Protein Expression Profiling in Cancer Cells
2
Immunohistochemical Analysis of ENO1 and Rab1A
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The paraffin-embedded tissues were immersed in boiling citrate buffer (Gene Tech, Shanghai, China, GT100202) for antigen retrieval, followed by a 15-mins incubation with 3% hydrogen peroxide (Yonghua Chemical Technology Co. LTD, Changshu China), and blocking with 5% FBS (Beyotime Inc, NanTong, China) for another 15 mins. The suitably treated sections were then incubated with primary antibodies against ENO1(A1033; 1:100 dilution; ABclonal; Wuhan, China) or Rab1A (Ab97956; 1:75 dilution; Abcam, Cambridge, MA, USA) at room temperature for 2–3 hrs and stained using a tissue staining kit (Zhongshan Biotechnology, Beijing, China) according to the manufacturer’s protocol. Five random high-power fields were observed per section, and the staining intensity was scored as 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong), and the percentage of positively stained cells as 1 (<25%), 2 (25–50%), 3 (51–75%), and 4 (>75%). The total score was calculated by multiplying the staining intensity score with the staining percentage score, and the samples were accordingly stratified into the low expression (− or +) and high expression (++ or +++) groups (0 = −; 1–4 = +; 5–8 =++; 9–12 = +++), as described in our previous study.18 (link)
3
VACV Infection in Cell Lines
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African green monkey kidney epithelioid cells (BS-C-1), and human cervix carcinoma epithelioid cells (HeLa) were grown in Dulbecco׳s modified Eagle׳s medium (DMEM) (Life Technologies) containing 50 IU/ml penicillin, 50 μg/ml streptomycin (Sigma) and 10% foetal bovine serum (FBS) (Life Technologies). Cells were incubated at 37 °C in a 5% CO2 incubator. VACV strain Western Reserve (VACV) and VACV-A5L-EGFP (Carter et al., 2003 (link)) were used in this work. The anti-A46 antibody was a kind gift from Andrew Bowie (Stack et al., 2005 (link)), the anti-D8 antibody a kind gift from Geoffrey Smith (Parkinson and Smith, 1994 (link)). Other antibodies used were anti-RAB1A (ab97956, Abcam), anti-GM130 (610822, BD Pharmingen), and anti-B5 (NR-556, BeiResources).
4
Antibody Panel for Autophagy Assays
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Primary antibodies used were as follows: rabbit anti‐human Tuj1 (Covance, IF: 1:500), mouse anti‐FLAG (M2, Sigma, WB: 1:2,000, IF: 1:1,000), mouse anti‐Myc (9B11, Cell Signaling, WB: 1:2,000, IF: 1:2,000), mouse anti‐tubulin (DM1A, Sigma, WB: 1:10,000), rabbit anti‐Myc (ab9106, Abcam, WB: 1:2,000, IF: 1:1,000), rabbit anti‐GAPDH (14C10, Cell Signaling, WB: 1:2,000), rabbit anti‐LC3 (2220, Novus Biologicals, WB: 1:1,000), mouse anti‐p62 (610833, BD Biosciences, IF: 1:1,000), rabbit anti‐p62 (18420‐1‐AP, ProteinTech, IF: 1:200), rabbit anti‐FIP200 (SAB4200135, Sigma, WB: 1:500), rabbit anti‐ULK1 (#8054, Cell Signaling, WB: 1:1,000), rabbit anti‐phospho‐ULK S757 (#6888, Cell Signaling, WB: 1:1,000), mouse anti‐EGFP (JL8, Clontech, WB: 1:5,000), rabbit anti‐ATG13 (#6940, Cell Signaling, WB: 1:1,000), rabbit anti‐Rab1a (Ab97956 Abcam, WB: 1:1,000), and rabbit anti‐HA (Sigma, WB: 1:2,000, IF: 1:1,000). Secondary antibodies used for immunoblotting were horseradish peroxidase‐coupled goat anti‐rabbit, goat anti‐rat, and rabbit anti‐mouse IgG (Dako; 1:5,000). Secondary antibodies used for immunofluorescence were Alexa fluorophore (488, 568, or 633)‐coupled goat/donkey anti‐mouse IgG, Alexa fluorophore (488, 568, or 633)‐coupled goat/donkey anti‐rabbit IgG (Invitrogen; 1:500).
5
Spinal Cord Protein Expression Analysis
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Proteins were extracted from the L2–L6 lumbar segment of the spinal cord, and their concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, China). Total proteins were separated in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20, incubated with specific primary antibodies overnight at 4°C, and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The primary antibodies used included anti-LC3-II/I (1 : 1000, 4108, CST), anti-Rab1 (1 : 1000, ab97956, Abcam), anti-autophagy-related protein 9 (ATG9, 1 : 1000, ab108338, Abcam), anti-P62 (1 : 2000, ab155686, Abcam), and anti-GAPDH (1 : 5000, 10494-1-AP, Proteintech). After three washes with PBS/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1 : 20000, PAB160011, BIO SWAMP). Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON) and analyzed using AlphaEase FC gel image analysis software.
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