Samples were diluted in SDS-PAGE Laemmli loading buffer (Bio-Rad) and electrophoresed on a 4–20% Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system. Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween 20) and then washed with PBST. In-house-made primary antibody, CR3022 as previously described14 (link),70 (approximate concentration 0.8–1.3 mg/mL), was added at a 1:10,000 dilution in PBST. Blots were washed with PBST, and secondary rabbit anti-human IgG H&L HRP (abcam ab6759) was added at 1:10,000 in PBST. Blots were developed using Pierce ECL substrate and imaged using a GE Amersham imager 600.
Rabbit anti human igg h l hrp
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-human IgG H&L HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to human immunoglobulin G (IgG) heavy and light chains in various immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx protein gel, by Bio-Rad (4 mentions)
Sds page laemmli loading buffer, by Bio-Rad (4 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Ettan dige imager, by GE Healthcare (1 mentions)
Imagequant software version 8, by GE Healthcare (1 mentions)
Anti icam 1 antibody, by Abcam (1 mentions)
Anti vcam 1 antibody, by Abcam (1 mentions)
Ripa buffer, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
P akt, by Abcam (1 mentions)
Lipofectamine 2000 kit, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Sybr premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
7 protocols using rabbit anti human igg h l hrp
1
Immunoblotting Assay for SARS-CoV-2 Antibody Detection
2
SARS-CoV-2 Antibody Western Blot Analysis
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Expi293F culture supernatants were collected
3 days after transfection,
harvested via spinning at 7000g for 15 min, and filtered
through a 0.22 μm filter. Samples were diluted in SDS-PAGE Laemmli
loading buffer (Bio-Rad), boiled at 95 °C, and run on a 4–20%
Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred
to nitrocellulose membranes using a Trans-Blot Turbo transfer system.
Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween
20) and then washed with PBST. In-house-made primary antibodies CR3022,
COVA2-15, and CB6 (approximate concentrations 0.8–1.3 mg/mL)
were added at a 1:10 000 dilution in PBST. Blots were washed
with PBST, and secondary rabbit antihuman IgG H&L HRP (abcam ab6759)
was added at 1:10 000 in PBST. Blots were developed using Pierce
ECL substrate and imaged using a GE Amersham imager 600.
3 days after transfection,
harvested via spinning at 7000g for 15 min, and filtered
through a 0.22 μm filter. Samples were diluted in SDS-PAGE Laemmli
loading buffer (Bio-Rad), boiled at 95 °C, and run on a 4–20%
Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred
to nitrocellulose membranes using a Trans-Blot Turbo transfer system.
Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween
20) and then washed with PBST. In-house-made primary antibodies CR3022,
COVA2-15, and CB6 (approximate concentrations 0.8–1.3 mg/mL)
were added at a 1:10 000 dilution in PBST. Blots were washed
with PBST, and secondary rabbit antihuman IgG H&L HRP (abcam ab6759)
was added at 1:10 000 in PBST. Blots were developed using Pierce
ECL substrate and imaged using a GE Amersham imager 600.
3
Peptide ELISPOT Protocol for Antibody Detection
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For peptide ELISPOT the following peptides were designed:
peptide #1 - RVLAPALDSWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #2 - LPKFSAPSASGPGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #3 - ESTRYQLWLPHQGGGDYKDDD{LYS(BIOTIN)} (Genescript)
control peptide - AVLAAALASWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
In brief, 110 pg biotin-conjugated peptides were printed on nitrocelluose coated slides (10485323, Whatman) by SpotBot® 4 (Arrayit). For primary antibody human precleared serum (1:100) was used, for secondary antibody rabbit anti-human IgG (H&L) (HRP) (Abcam) was used. All incubations were done for 1 h at room temperature. Results were scanned using Ettan DigeImager (GE Healthcare Life Sciences) and images calculated using ImageQuant software version 8.1 (GE Healthcare Life Sciences).
peptide #1 - RVLAPALDSWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #2 - LPKFSAPSASGPGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #3 - ESTRYQLWLPHQGGGDYKDDD{LYS(BIOTIN)} (Genescript)
control peptide - AVLAAALASWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
In brief, 110 pg biotin-conjugated peptides were printed on nitrocelluose coated slides (10485323, Whatman) by SpotBot® 4 (Arrayit). For primary antibody human precleared serum (1:100) was used, for secondary antibody rabbit anti-human IgG (H&L) (HRP) (Abcam) was used. All incubations were done for 1 h at room temperature. Results were scanned using Ettan DigeImager (GE Healthcare Life Sciences) and images calculated using ImageQuant software version 8.1 (GE Healthcare Life Sciences).
4
Protein Extraction and Western Blot Analysis
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Total protein extracted from cells using RIPA buffer (Abcam) was quantified with a BCA Kit (Beyotime). Western blot analysis was carried out as previously reported.26 All antibodies used in this study were purchased from Abcam: Anti‐BMP4 antibody (ab235114), Anti‐ICAM1 antibody (ab171123), Anti‐VCAM1 antibody (ab134047), Anti‐VEGFA (ab46154), Anti‐Ki67 (ab16667), Anti‐CD31 (ab9498), Anti‐GAPDH antibody (ab8245), and Rabbit Anti‐Human IgG H&L (HRP) (ab6759).
5
Immunoblotting Assay for SARS-CoV-2 Antibody Detection
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Samples were diluted in SDS-PAGE Laemmli loading buffer (Bio-Rad) and electrophoresed on a 4–20% Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system. Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween 20) and then washed with PBST. In-house-made primary antibody, CR3022 as previously described14 (link),70 (approximate concentration 0.8–1.3 mg/mL), was added at a 1:10,000 dilution in PBST. Blots were washed with PBST, and secondary rabbit anti-human IgG H&L HRP (abcam ab6759) was added at 1:10,000 in PBST. Blots were developed using Pierce ECL substrate and imaged using a GE Amersham imager 600.
6
Immunoblotting Assay for SARS-CoV-2 Antibody Detection
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Samples were diluted in SDS-PAGE Laemmli loading buffer (Bio-Rad) and electrophoresed on a 4–20% Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system. Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween 20) and then washed with PBST. In-house-made primary antibody, CR3022 as previously described14 (link),70 (approximate concentration 0.8–1.3 mg/mL), was added at a 1:10,000 dilution in PBST. Blots were washed with PBST, and secondary rabbit anti-human IgG H&L HRP (abcam ab6759) was added at 1:10,000 in PBST. Blots were developed using Pierce ECL substrate and imaged using a GE Amersham imager 600.
7
Breast Cancer Cell Line Analysis
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Breast cancer cells MCF-7 and MDA-MB-231 were purchased from ATCC in the United States. DMEM medium and RPMI-1640 medium were from Gibco Corporation, the United States. EpCAM Nglycosylation mutant plasmids, empty vector control plasmids, and strains were purchased from Guangzhou RiboBio Co., Ltd. Lipofectamine 2000 kits were from Thermo Fisher Scientific Inc., the United States. PrimeScript™ RT reagent Kits with gDNA Eraser (Perfect Real Time) kits and SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus) kits were produced by Takara Bio (Beijing) Inc. Rabbit monoclonal antibody EpCAM, rabbit monoclonal Ecadherin, rabbit monoclonal N-cadherin, rabbit monoclonal Vimentin, rabbit monoclonal Caspase-3, rabbit monoclonal Bcl-2, rabbit monoclonal Bax, rabbit monoclonal p38, rabbit monoclonal p-p38, rabbit monoclonal PI3K, rabbit monoclonal p-PI3K, rabbit monoclonal Akt, rabbit polyclonal antibody p-Akt, primary protein antibody of mouse monoclonal β-actin, and the secondary antibody of rabbit antihuman IgG H&L (HRP) were produced by Abcam PLC., the United Kingdom.
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