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Immuno blot

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The Immuno-Blot is a laboratory instrument used for protein analysis and detection. It is designed to perform Western blotting, a widely used technique in molecular biology and biochemistry. The core function of the Immuno-Blot is to separate and transfer proteins from a gel to a membrane, allowing for the detection and analysis of specific proteins using antibodies.

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Lab products found in correlation

Mmp 13, by Santa Cruz Biotechnology (2 mentions) Molecular imager chemidoc xrs system, by Bio-Rad (2 mentions) Ripa buffer, by Solarbio (2 mentions) Semi dry transfer, by Bio-Rad (1 mentions) Chemilmagertm 4400, by Bio-Techne (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Ecl chemiluminescence kit, by Santa Cruz Biotechnology (1 mentions) Difco skim milk, by BD (1 mentions) Bcatm protein assay kit, by Beyotime (1 mentions) Col2a1, by Proteintech (1 mentions) Hoxa3, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Protein assay, by Bio-Rad (1 mentions) Cytochrome c, by Abcam (1 mentions) Ecl western blotting substrate, by Solarbio (1 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Col2a1, by Santa Cruz Biotechnology (1 mentions) Silver staining, by Bio-Rad (1 mentions) Clarity ecl reagent, by Bio-Rad (1 mentions) Tris glycine sds page gel, by Bio-Rad (1 mentions)

18 protocols using immuno blot

1

Antigenicity and Immunogenicity Analysis

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Example 5

In order to determine the antigenicity and immunogenicity, Western blot using anti-diphtheria toxin (DT) of each sample obtained in each purification step was performed, and the result is shown in FIG. 6.

Each sample obtained in each purification step was developed by SDS-PAGE (FIG. 6(a)). Then, the protein was adsorbed onto Immuno-blot poly-vinyl difluoride (PVDF) membrane using an instrument (semi-dry transfer, Biorad), and the analysis was achieved by Western blot method with mouse monoclonal anti-diphtheria toxin as primary antibody and goat polyclonal anti-mouse-IgG-HRP as secondary antibody (FIG. 5(b)). In Lanes 1 to 3, the weak signals (WB signals) are considered to be due to the reduction in membrane transfer efficiency above 100 kD. An unspecific band was noted in the unpurified sample, but the binding with CRM197 was clearly noted as the purity of the sample increased.

US11124548B2. Method for expressing and purifying soluble protein of CRM197 (2021-09-21). FORBIOKOREA CO., LTD. [KR]. Inventors: Hyeon Cheol Lee [KR], Bong Seong Koo [KR], Hyoung Jong Seo [KR], Jin Sook Kim [KR], Ah Reum Park [KR], Seung Won Jang [KR].
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2

Whole Cell Lysis and Immunoblotting

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Whole cell lysates were prepared on ice with RIPA buffer containing protease inhibitor cocktail (Roche) as described [21 (link)]. The lysates were centrifuged at 16,000× g at 4 °C for 10 min, and the supernatants were collected as whole cell lysates. Whole cell lysates were separated on a SDS-polyacrylamide gel and blotted onto a PVDF membrane (Immuno-Blot, Bio-Rad). The antigen-antibody complexes were visualized using the chemiluminescence substrate SuperSignal®, West Pico (Pierce). For quantitative analysis, respective bands were quantified using a ChemilmagerTM 4400 (Alpha Innotech Corporation, San Leandro, CA, USA).
Vogel C.F., Ishihara Y., Campbell C.E., Kado S.Y., Nguyen-Chi A., Sweeney C., Pollet M., Haarmann-Stemmann T, & Tuscano J.M. (2019). A Protective Role of Aryl Hydrocarbon Receptor Repressor in Inflammation and Tumor Growth. Cancers, 11(5), 589.
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3

Western Blot Analysis of APP Phosphorylation

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In Western blot (WB) experiments, 30 µg of total protein HELA cells and 60 µg of total protein from blood mononuclear cell (PBMCs) of patients were loaded onto 8% polyacrylamide gel after being denatured at 95 °C for 5 min. Total protein concentration was determined by the Bradford method, using bovine serum albumin (BSA) as standard. For APP Tyr682 phosphorylation detection, 100 μg of total lysates from HELA cells was incubated overnight with phospho-Tyrosine Mouse mAb Magnetic Bead Conjugate (P-Tyr-100) (Cell Signaling, #8095; Milan, Italy). Immunoprecipitated (IP) samples were then analysed by WB. Gels were transferred to PVDF membranes (Immuno-Blot; Bio Rad, Segrate, MI, Italy) and incubated in primary rabbit anti APP antibody clone Y188 (Abcam, ab32136, Cambridge, UK) antibody and secondary monoclonal anti-β-actin-peroxidase (Merk Life Science S.r.l. MI, Italy) antibody.
Reveglia P., Nasso R., Angiolillo A., Lecce L., Paolillo C., De Tullio S., Gelzo M., Di Costanzo A., Matrone C, & Corso G. (2021). Tandem Mass Spectrometry as Strategy for the Selective Identification and Quantification of the Amyloid Precursor Protein Tyr682 Residue Phosphorylation Status in Human Blood Mononuclear Cells. Biomolecules, 11(9), 1297.
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4

Western Blot Analysis of Cellular Proteins

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The total protein of cells was extracted using RIPA buffer (Solarbio Biotech, Beijing, China). Protein concentrations were examined using BCATM protein assay kit (Beyotime Biotechnology, Shanghai, China). A total of 30ug protein was loaded onto the PAGE (EpiZyme, Shanghai, China), separated by electrophoresis and then transferred onto PVDF membranes (Immunoblot, Bio-Rad). The membranes were blocked with 5% non-fat milk (Difco™ Skim Milk, BD) for 1 h at room temperature and incubated with primary antibodies against COL2A1 (Proteintech, Wuhan, China), MMP13(Santa Cruz, UK), BAX(CST, USA), HOXA3(Santa Cruz, USA), GAPDH(CST, USA) at 4 °C overnight. Next, the membranes were incubated with secondary antibodies(Santa Cruz, USA) for 1 h at room temperature. The protein signaling were visualized with ECL chemiluminescence kit(Santa Cruz Biotechnology, Dallas, TX, USA) using Molecular Imager ChemiDoc XRS System(Bio-Rad).
Li H.Z., Xu X.H., Lin N., Wang D.W., Lin Y.M., Su Z.Z, & Lu H.D. (2020). Overexpression of miR-10a-5p facilitates the progression of osteoarthritis. Aging (Albany NY), 12(7), 5948-5976.
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5

Whole Cell Lysate Preparation and Western Blotting

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In order to prepare whole cell lysates, cells were lysed on ice with RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich) for 30 min. The lysates were centrifuged at 16,000 g at 4°C for 10 min, and the supernatants were collected as whole cell lysates. Protein concentration in the whole cell lysate was determined by BIO-RAD protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Whole cell lysates were separated on 8% SDS-polyacrylamide gel and blotted onto a PVDF membrane (Immuno-Blot, Bio-Rad). The antigen-antibody complexes were visualized using the chemiluminescence substrate SuperSignal®, West Pico (Pierce, Rockford, IL) as recommended by the manufacture. For quantitative analysis, respective bands were quantified using a ChemilmagerTM 4400 (Alpha Innotech Corporation, San Leandro, CA).
Bekki K., Vogel H., Li W., Ito T., Sweeney C., Haarmann-Stemmann T., Matsumura F, & Vogel C.F. (2014). The aryl hydrocarbon receptor (AhR) mediates resistance to apoptosis induced in breast cancer cells. Pesticide biochemistry and physiology, 120, 5-13.
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6

Chondrocyte Protein Isolation and Western Blot Analysis

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For isolation of cell proteins, HC‐a chondrocytes were washed with cold PBS twice and treated with RIPA buffer (Solarbio Biotech, Beijing, China). Cell lysates containing an equal amount of proteins were separated by electrophoresis and then transferred onto PVDF membranes (Immunoblot, Bio‐Rad). The membranes were blocked in TBST containing 5% non‐fat milk (Difco™ Skim Milk, BD) for 1 h at room temperature. After blocking, the membranes were incubated with primary antibodies against COL2A1 (Santa Cruz, USA), MMP13 (Santa Cruz, USA), PON2 (Abcam, USA), BCL2 (CST, USA), BAX (CST, USA), Cytochrome C (Abcam, USA), GAPDH (CST, USA) at 4 °C overnight, and then incubated with horseradish peroxidase(HRP)‐conjugated secondary antibody (Santa Cruz, USA). The antibody binding was detected using an ECL Western Blotting Substrate (Solarbio Science & Technology) and visualized using a Molecular Imager ChemiDoc XRS System (Bio‐Rad).
Shang J., Li H., Wu B., Jiang N., Wang B., Wang D., Zhong J., Chen Y., Xu X, & Lu H. (2022). CircHIPK3 prevents chondrocyte apoptosis and cartilage degradation by sponging miR‐30a‐3p and promoting PON2. Cell Proliferation, 55(9), e13285.
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7

Oligomer Analysis of MaSAMP Peptides

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For SAMP and SAMP-helix2 oligomers analyses, 1 μM solutions of MaSAMP and MaSAMP-helix2 were made by diluting 1 μL 120 ng MaSAMP or 360 ng MaSAMP-helix2 peptide stock (in DMSO) in 20 μL sample buffer (100 mM Tris HCl, pH6.8, 10% glycerol, 0.1 M DTT, and 0.0006% Bromophenol blue) in a time series and resolved in a 15% PAGE gel without SDS. The oligomers of MaSAMP or MaSAMP-helix2 were detected by immunoblot or silver staining (following the manufacturer’s instructions, Bio-Rad), respectively.
Huang C.Y., Araujo K., Sánchez J.N., Kund G., Trumble J., Roper C., Godfrey K.E, & Jin H. (2021). A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. Proceedings of the National Academy of Sciences of the United States of America, 118(6), e2019628118.
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8

Whole Cell Lysate Preparation and Western Blot

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Total cell lysates were prepared as follows. Cells after treatment were washed with phosphate-buffered saline (PBS) and trypsinized, pelleted and stored at −80°C. Frozen cell pellets were lysed using urea lysis buffer [8 M urea, 50 mM Tris-HCl (pH 8.0), 75 mM NaCl, 1 mM NaF, 1 mM NaV, 1 mM β-glycerophosphate and 25 mM NEM]. Lysates were quantified using a BCA assay (Pierce). Total protein (25 μg) was resolved on 4–20% Tris-glycine SDS–PAGE gels (Bio-Rad). Resolved proteins were blotted to PVDF membranes (Bio-Rad Immuno-Blot) using Bjerrum semi-dry transfer buffer [48 mM Tris, 39 mM glycine, 0.0375% (w/v) SDS, 20% (v/v) methanol, pH 9.2) in a Bio-Rad Trans-Blot Turbo transfer apparatus at 30V constant for 30 mins. Blots were incubated with relevant primary and HRP-conjugated secondary antibodies and were detected using Clarity ECL reagent (Bio-Rad) on a Bio-Rad ChemiDoc XRS+ system.
Sundaramoorthy E., Ryan A.P., Fulzele A., Leonard M., Daugherty M.D, & Bennett E.J. (2021). Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection. Journal of Cell Science, 134(8), jcs257188.
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9

Immunoblotting Protocol for Protein Analysis

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For immunoblot (Bio-Rad, America), cells were lysed with RIPA lysis buffer for total protein. Samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After the blockage with 5% nonfat milk, membranes were then incubated with Anti-Sortilin, Mouse monoclonal to CML (Abcam Cat# ab125145, RRID: AB_11127913), Rabbit monoclonal to CD9 (Abcam Cat# ab92726, RRID: AB_10561589), Mouse monoclonal to beta Actin (Abcam Cat# ab8226, RRID: AB_306371), Rabbit polyclonal to Runx2 antibody (Abcam Cat# ab23981, RRID: AB_777785) overnight at 4°C. Then the membranes were washed with TBST, and the diluted secondary antibody (1:5000) was added and incubated at 37°C for 1.5 hours. After enhancement with the ECL detection kit, analysis was performed with a gel imaging system (Amersham Imager 600).
Jing L., Li L., Ren X., Sun Z., Bao Z., Yuan G., Cai H., Wang L., Shao C, & Wang Z. (2020). Role of Sortilin and Matrix Vesicles in Nε-Carboxymethyl-Lysine-Induced Diabetic Atherosclerotic Calcification. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 13, 4141-4151.
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10

Whole Cell Protein Lysate Immunoblotting

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Whole cell protein lysate (60 μg/sample) was used. Samples were run on denaturing sodium dodecylsulfate-polyacrylamide gel electrophoresis gels (12%) before transferring onto Immuno-Blot™ polyvinylidene fluoride membranes (Bio-Rad). The reference protein β-actin was used as a loading control.
Li Y., Lim C., Dismuke T., Malawsky D.S., Oasa S., Bruce Z.C., Offenhäuser C., Baumgartner U., D’Souza R.C., Edwards S.L., French J.D., Ock L.S., Nair S., Sivakumaran H., Harris L., Tikunov A.P., Hwang D., Del Mar Alicea Pauneto C., Maybury M., Hassall T., Wainwright B., Kesari S., Stein G., Piper M., Johns T.G., Sokolsky-Papkov M., Terenius L., Vukojević V., Gershon T.R, & Day B.W. (2023). Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179. Research Square.
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