Western blotting was performed to measure protein changes. In brief, the samples (tissue and cell samples) were lysed in RIPA buffer (P0013C, Beyotime, Guangzhou, China) and mixed with sample buffer (No. 1610737, Bio–Rad) at a 1:1 ratio. Equal amounts of protein samples were separated by 10% SDS–PAGE and blotted onto PVDF membranes after transmembrane electrophoresis. The antibodies used included Runx1 (No. 39000, ChIP grade, Active motif, Carlsbad, CA) and FLAG (ab125243, Abcam, Cambridge, UK and 9A3, Cell Signaling Technology, Boston, MA, 1:1 000 dilution). The secondary antibodies included mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Biotech, Delaware Avenue, CA, 1:2 000 dilution) and m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotech, 1:2 000 dilution).
Mouse anti rabbit igg hrp sc 2357
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-rabbit IgG-HRP (sc-2357) is a horseradish peroxidase (HRP)-conjugated secondary antibody that binds to rabbit immunoglobulin G (IgG). It is designed for use in Western blotting, ELISA, and other immunoassay applications to detect and quantify rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
M iggκ bp hrp sc 516102, by Santa Cruz Biotechnology (3 mentions)
M iggk bp hrp sc 516102, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Beyotime (1 mentions)
Sample buffer, by Bio-Rad (1 mentions)
Tmb stop solution, by LGC (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Ecl western blotting detection system, by Cytiva (1 mentions)
Hybond ecl, by Cytiva (1 mentions)
Anti gapdh g9545, by Merck Group (1 mentions)
Anti actin ab3280, by Abcam (1 mentions)
Hybond, by Cytiva (1 mentions)
Immobilon crescendo western hrp substrate, by Merck Group (1 mentions)
Ab109201, by Abcam (1 mentions)
Ab118307, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab92726, by Abcam (1 mentions)
Mini gel apparatus, by Bio-Rad (1 mentions)
Sc 7148, by Santa Cruz Biotechnology (1 mentions)
Ab1791, by Abcam (1 mentions)
P iκbα, by Thermo Fisher Scientific (1 mentions)
16 protocols using mouse anti rabbit igg hrp sc 2357
1
Quantifying Protein Levels via Western Blot
2
Influenza Microneutralization Assay
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Influenza microneutralization assay was performed as previously described with modifications (Grund et al., 2011 (link)). In brief, sera samples were first treated with RDE II and heated inactivated, as described in the HAI assay above. Then, sera were serially diluted with serum‐free DMEM (Merck) supplemented with penicillin/streptomycin, L‐glutamine and 1 μg/ml TPCK‐Trypsin, and incubated with X31 virus for 1 h at 37°C. Following incubation, the sera‐virus mixture was transferred to preseeded MDCK cell monolayer which was washed with serum‐free DMEM, and cultured at 37°C. Twenty‐four hours later, cells were washed with PBS, fixed with 80% ice‐cold acetone and the infection was quantified by an influenza nucleoprotein ELISA. Plates were first blocked with 5% non‐fat milk in PBST, and then sequentially incubated with rabbit anti‐NP antibody (PA5‐32242; Thermo Fisher Scientific) and mouse anti‐rabbit IgG‐HRP (sc2357; Santa Cruz). After extensive washes, plates were developed with TMB solution (Sera Care) for 5 min at RT and stopped with TMB stop solution (Sera Care). Plates were finally read at a testing wavelength of 450 nm and a reference wavelength of 800 nm, and the IC50 was calculated for each sample.
3
Western Blotting Technique for Protein Analysis
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5 μg of protein per lane was separated on 10% SDS/PAGE gel and transferred onto a Hybond ECL® (enhanced chemiluminescence) membrane (Amersham Biosciences). After blocking with 5% milk for 1 h, the membrane was incubated overnight at 4°C with primary antibodies diluted 1:1000 followed by a further 1 h incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (Bio-Rad) at a 1:10000 dilution, and the signal was detected with the ECL® Western blotting detection system (Amersham Biosciences). The following primary antibodies were used for immunoblotting: Anti-AKAP1 (D9C5), Anti-COX4 (3E11), Anti-Mios (D12C6), Anti-Sestrin2 (D186) were from Cell Signaling Technology; Anti-Actin (ab3280) was from Abcam; Anti-GAPDH (G9545) was from Sigma-Aldrich. Secondary antibodies were from Santa Cruz Biotechnology: m-IgGκ BP-HRP (sc-516102) and mouse anti-rabbit IgG-HRP (sc-2357).
4
Extracellular Vesicle Protein Analysis
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EVs derived from MSCs were isolated through centrifugation, and the amount of total protein was quantified using the Bradford assay. Protein samples were separated using 10% SDS-PAGE with a mini gel apparatus (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (T831.1, Merck Millipore, MA, USA). Each membrane was blocked with 3% skim milk in Tris-buffered saline containing 0.05% Tween 20. The primary antibodies anti-CD9 (1:1000, ab92726, Abcam, MA, USA), CD63 (1:1000, ab118307, Abcam, MA, USA), and anti-CD81 (1:1000, ab109201, Abcam, MA, USA) were used as primary antibodies and were incubated overnight at 4C. Mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz, CA, USA) was used as a secondary antibody for 1h. The bands were visualized using enhanced chemiluminescence according to the manufacturers instructions (Immobilon Crescendo Western HRP substrate, Millipore, Darmstadt, Germany). The band intensities were quantified using the ChemiDoc XRS+System (Bio-Rad). All the samples were developed within 10min to obtain a band.
5
Western Blot Analysis of LANA and Apoptosis Markers
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Ex vivo treated PEL cells were solubilized in lysis buffer. One hundred µg of protein lysates were loaded onto a 12% SDS-polyacrylamide gel, subjected to electrophoresis, and transferred onto nitrocellulose membranes. Blots were incubated overnight with specific primary antibodies against LANA-1 (NBP1-30176, Novus), LANA-2 (NB200-167H, Novus), Caspase 3 (sc-7148, Santa Cruz), PARP (sc-7150, Santa Cruz), p-IκBα (Invitrogen, Foster City, CA, USA)., H3 total (ab1791, Abcam, Cambridge, UK), and GAPDH (MAB5476, Abnova, Walnut, CA, USA). Blots were then incubated with appropriate HRP-conjugated secondary antibodies (m-IgGK BP-HRP sc-516102, mouse anti-rabbit IgG-HRP sc-2357, Santa Cruz, CA, USA). Bands were visualized by chemiluminescence (Clarity max, Bio-Rad, Hercules, CA, USA Cat# 170-5061).
6
Comprehensive Antibody Validation Protocol
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The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): anti-OGT (sc-74546), anti-E6AP (sc-166689), anti-hemagglutinin (HA) (sc-7392), anti-actin (sc-8432), anti-UB (sc-8017), and mouse anti-rabbit IgG-HRP (sc-2357). Goat anti-mouse IgG secondary antibody (31438) was from Thermo Fisher Scientific (Waltham, MA, USA). Anti-FLAG (M2) antibody (F3165) was from Sigma-Aldrich (Burlington, MA, USA). Anti-Nup62 antibody (NBP1-31381) was from Novus Biologicals (Centennial, CO, USA). Anti-Sp1 antibody (07-645) was from EMD Millipore (Burlington, MA, USA). Anti-O-GlcNAc RL2 antibody (2739) was from Abcam (Cambridge, MA, USA). E6 protein of human papillomavirus type 16 was from Boston Biochem (Cambridge, MA, USA).
7
Quantification of KIF2A and Ki-67 in Tumors
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In situ expression of KIF2A and proliferation marker ki-67 in human tumours and xenograft mice tumours was measured using IHC method. Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded tissues were sliced into 4 µm slides. Antigen retrieval was performed using 0.01 M citrate buffer for 20 min at 95 °C, and slides were incubated in 3% H2O2 solution for 15 min at 25 °C. Blocked with 5% goat serum for 30 min incubation at 25 °C, the slides were incubated with primary antibodies overnight at 4 °C and secondary antibody for 30 min at 25 °C, and re-stained with diaminobenzidine and haematoxylin; eventually, tissue protein expression of KIF2A and ki-67 was observed under microscope (Nikon Microsystems, Shanghai, China) at an appropriate magnification (100×). Anti-KIF2A (sc-398010, 1:100) and anti-ki-67 (ARG53222; 1:200) were from Santa Cruz Biotechnology and arigo Biolaboratories (Taiwan, China), respectively. The m-IgGκ BP-HRP (sc-516102; 1:100) and mouse anti-rabbit IgG-HRP (sc-2357; 1:100) were from Santa Cruz Biotechnology (Shanghai, China).
8
Investigating Gastric Cancer Cell Lines
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Human gastric cancer cell lines SGC-7901 and BGC-823 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a humidified incubator with an atmosphere of 5% CO2. N-acetyl-L-cysteine (NAC), L-glutathione (GSH) and cisplatin were purchased from Sigma (St. Louis, MO, USA). BMS-582949 and SP600125 were purchased from Selleck Chemicals (Houston, TX, USA). WZ35 was synthesized in our laboratory as previously described [13 (link)]. Antibodies including anti-Bcl-2 (sc-7382, 1:50), anti-TrxR1 (sc-28,321, 1:200), anti-GAPDH (sc-47,724, 1:200), mouse anti-rabbit IgG-HRP (sc-2357, 1:2000) and m-IgGκ BP-HRP (sc-516,102, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies including anti-p-p38 (4631, 1:1000), anti-p38 (9212, 1:1000), anti-p-JNK (4668, 1:1000) and anti-JNK (9252, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-Ki-67 (ab15580, 1:1000) antibody was purchased from Abcam (Cambridge, MA, USA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).
9
Antibody Validation for Protein Analysis
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Antibodies specific for DYKDDDDK (FLAG tag, #14793) and γH2AX (#80312) and mouse IgG‐HRP (#7076) were purchased from Cell Signaling Technology, USA. Antibodies specific for GAPDH (sc‐47724) and HPV16‐E7 (sc‐6981) and mouse antirabbit IgG‐HRP (sc‐2357) were purchased from Santa Cruz Biotechnology, USA. Antibodies specific for HPV16/18‐E6 (ab70) were purchased from Abcam, USA. Antibodies specific for SERPINB3 (26558‐1‐AP), USP1 (14346‐1‐AP), FANCD2 (28619‐1‐AP), FANCI (67304‐1‐Ig), and KI67 (27309‐1‐AP) were purchased from Proteintech, China.
Chemicals were purchased from Sigma. All of the culture media (DMEM, DMEM‐F12) and fetal bovine serum (FBS) were purchased from Biological Industries, China.
Chemicals were purchased from Sigma. All of the culture media (DMEM, DMEM‐F12) and fetal bovine serum (FBS) were purchased from Biological Industries, China.
10
Western Blot Antibody Inventory
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Antibodies specific for DYKDDDDK (FLAG tag) (#14,793), LCN2 (#44,058), p-EGFR (Tyr1173, #4407), ERK (#4696), p-ERK (#4370) and mouse IgG-HRP (#7076) were purchased from Cell Signaling Technology. Antibodies specific for GAPDH (sc-47724) and β-tubulin (sc-166729) and mouse anti-rabbit IgG-HRP (sc-2357) were purchased from Santa Cruz Biotechnology. Antibodies specific for ERK1/2 (YT1625) and p-ERK1/2 (YP0101) were purchased from ImmunoWay. Antibodies specific for LCN2 (26,991–1-AP), EGFR (66,455–1-Ig, 51,071–2-AP), and KI67 (27,309–1-AP) were purchased from Proteintech.
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