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Reducing sample buffer

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Reducing sample buffer is a laboratory reagent used to prepare samples for analysis. It is designed to denature and reduce proteins, allowing for better separation and detection during subsequent analytical procedures.

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Lab products found in correlation

β actin, by Abcam (5 mentions) Horseradish peroxidase conjugated goat anti mouse and goat anti rabbit secondary antibodies, by Agilent Technologies (5 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)

6 protocols using reducing sample buffer

1

CLDN6 Protein Expression in Cancers

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Example 2

For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N(SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

US10919974B2. Antibodies for treatment of cancer expressing claudin 6 (2021-02-16). Ganymed Pharmaceuticals AG [DE], Johannes Gutenberg-Universitat Mainz [DE]. Inventors: Ugur Sahin [DE], Ozlem Tureci [DE], Michael Koslowski [DE], Korden Walter [DE], Stefan Woll [DE], Maria Kreuzberg [DE], Bernd Hubner [DE], Michael Erdeljan [DE], Michael Weichel [DE].
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2

Western Blot Analysis of CLDN6 Expression

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Example 2

For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N(SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

US09932401B2. Antibodies specific for claudin 6 (CLDN6) (2018-04-03). Ganymed Pharmaceuticals AG [DE], Johannes Gutenberg-Universitat Mainz [DE]. Inventors: Ugur Sahin [DE], Özlem Türeci [DE], Michael Koslowski [DE], Korden Walter [DE], Stefan Woll [DE], Maria Kreuzberg [DE], Bernd Hubner [DE], Michael Erdeljan [DE].
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3

Protein Expression Analysis of CLDN6

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Example 2

For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N (SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

US11859008B2. Antibodies for treatment of cancer expressing claudin 6 (2024-01-02). Ganymed Pharmaceuticals GmbH [DE], Johannes Gutenberg-Universitat Mainz [DE]. Inventors: Ugur Sahin [DE], Ozlem Tureci [DE], Michael Koslowski [DE], Korden Walter [DE], Stefan Woll [DE], Maria Kreuzberg [DE], Bernd Hubner [DE], Michael Erdeljan [DE], Michael Weichel [DE].
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4

Quantifying CLDN6 Protein Expression

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Example 2

For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N(SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

US11858988B2. Antibodies specific for claudin 6 (CLDN6) (2024-01-02). Ganymed Pharmaceuticals GmbH [DE], Johannes Gutenberg-Universitat Mainz [DE]. Inventors: Ugur Sahin [DE], Özlem Türeci [DE], Michael Koslowski [DE], Korden Walter [DE], Stefan Woll [DE], Maria Kreuzberg [DE], Bernd Hubner [DE], Michael Erdeljan [DE].
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5

Western Blot Analysis of CLDN6 Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 2

For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N(SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

US10745477B2. Antibodies specific for claudin 6 (CLDN6) (2020-08-18). Ganymed Pharmaceuticals AG [DE], Johannes Gutenberg-Universitat Mainz [DE]. Inventors: Ugur Sahin [DE], Özlem Türeci [DE], Michael Koslowski [DE], Korden Walter [DE], Stefan Woll [DE], Maria Kreuzberg [DE], Bernd Hubner [DE], Michael Erdeljan [DE].
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6

Western Blot Protein Analysis Protocol

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Samples were lysed in a buffer containing 50 mM Tris, 1% Triton-X 100, 131 mM sodium chloride, 1 mM sodium diphosphate, 1 mM sodium fluoride, 1 mM EDTA, 1% protease inhibitor, and 1% phosphatase inhibitor with a homogenizator for 10 min and subsequently centrifuged at 4°C with 16,000 rpm for 10 min. The supernatant was collected, and quantification of the protein concentration was photometrically accomplished (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific). Reducing sample buffer (Carl Roth, Karlsruhe, Germany) was added, and the samples were heated for 5 min at 95°C. Equal amounts of protein were separated on 8–12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, California, United States). Following transfer, the membranes were blocked for 1 h and incubated with the primary antibodies against ABCB1 (Abcam, 0.5 μg/ml), NF-κB p65 (Abcam, 0.5 μg/ml), MMP-9 (Abcam, 0.5 μg/ml), β-actin (0.2 μg/ml), and α-tubulin (0.1 μg/ml) overnight. After washing with tris-buffered saline supplemented with 0.1% Tween 20® detergent (TBS-T) three times, the blots were incubated with horseradish peroxidase coupled secondary anti-mouse-antibody and anti-rabbit-antibody (1:10,000) for 1 h. The membranes were bathed in ECL reagent and developed with the imaging system ChemiDocTM XRS + (Bio-Rad).
Janssen L., Ai X., Zheng X., Wei W., Caglayan A.B., Kilic E., Wang Y.C., Hermann D.M., Venkataramani V., Bähr M, & Doeppner T.R. (2021). Inhibition of Fatty Acid Synthesis Aggravates Brain Injury, Reduces Blood-Brain Barrier Integrity and Impairs Neurological Recovery in a Murine Stroke Model. Frontiers in Cellular Neuroscience, 15, 733973.
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