Wet blot transfersystem

Manufactured by Avantor

The wet blot transfer system is a laboratory equipment used for the transfer of proteins from a gel to a membrane, such as nitrocellulose or PVDF, for further analysis. This system uses an electric current to facilitate the transfer, ensuring efficient and consistent results.

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Lab products found in correlation

Westernbright chemiluminescent substrate, by Advansta (1 mentions) Anti gapdh, by Enzo Life Sciences (1 mentions) Anti hsp90β, by Enzo Life Sciences (1 mentions) Imagequant 800, by Cytiva (1 mentions) Amersham imagequant, by Cytiva (1 mentions)

2 protocols using wet blot transfersystem

Protein Expression Analysis in HeLa Cells

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HeLa cells (1.5 × 10⁶) were treated
with CNC and GNC (0, 200, 300, 400, and 500 μg/mL)
for 48 h. Proteins were extracted from the cells in the lysis buffer
(20 mM Tris–HCl pH 7.4, 2 mM EDTA, 150 mM NaCl, 1.2% sodium
deoxycholate, 1.2% TritonX-100, protease inhibitor cocktail (PIC))
and quantified by the Bradford method. An equivalent amount of proteins
(75 μg) were resolved by 15% SDS-PAGE and transferred onto a
nitrocellulose membrane (GVS Life Science) with a wet blot transfer
system (VWR). The membranes were blocked with 2–5% nonfat milk
in TBS-Tween 20 (0.2%) and incubated with primary antibodies (see Table S1 for reagent details) with the following
dilutions: anti-Caspase 9 (1:1000) and anti-α-tubulin (1:5000).
Lysates (50 μg) of HFs-hTERT cells and their transformed variant
were resolved by 10% SDS-PAGE, processed as described above, and anti-H-Ras
(1:1000) and anti-SV40 LT (1:750) antibodies were used to detect the
corresponding proteins. Then membranes were washed with TBS-Tween
20 (0.2%) and incubated with the appropriate HRP-conjugated secondary
antibodies (1:10,000 dilutions). Immunoblots were developed using
the WesternBright chemiluminescent substrate (Advansta). Images were
recorded with a LI-COR Odyssey image recorder.

Bhattacharya S.R., Bhattacharya K., Xavier V.J., Ziarati A., Picard D, & Bürgi T. (2022). The Atomically Precise Gold/Captopril Nanocluster Au25(Capt)18 Gains Anticancer Activity by Inhibiting Mitochondrial Oxidative Phosphorylation. ACS Applied Materials & Interfaces, 14(26), 29521-29536.

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Immunoblotting for HSP Proteins

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Lysates of cells or mouse tissues (20–100 μg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (GVS Life Science) with a wet blot transfer system (VWR). Membranes were blocked with 2–5% non-fat milk or BSA in TBS-Tween 20 (0.2%) and incubated with primary antibodies with the following dilutions: anti-Hsp90α (1:2000; polyclonal antiserum from Synaptic Systems and monoclonal antibodies from Enzo Lifesciences), anti-Hsp90β (1:2000), anti-Hop (1:1000), anti-GAPDH (1:7500), anti-β-actin (1:5000), anti-Hsp70 (1:2000), anti-Hsc70 (1:2000), anti-c-Raf (1:1000), anti-Ub (1:5000), anti-p23 (1:1000), anti-Cdc37 (1:1000), anti-Akt (1:1000), anti-Hsf1 (1:1000), anti-Hsp40/Hdj1 (1:1000), anti-Hsp110 (1:1000), anti-Aha1 (1:2000), anti-Hsp25/27 (1:2000), anti-Puromycin (1:22,000), anti-p-mTOR (1:1000), anti-mTOR (1:2000), anti-p-S6 (1:1000), anti-p-eIF2α (1:1000), anti-eIF2α (1:1000). Membranes were washed with TBS-Tween 20 (0.2%) and incubated with the corresponding secondary antibodies: anti-mouse IgG-HRP (1:10,000), anti-rabbit IgG-HRP (1:10,000), and anti-rat IgG-HRP (1:10,000). Immunoblots were developed using the WesternBright^TM chemiluminescent substrate (Advansta). Images were recorded by using a LI-COR Odyssey or Amersham ImageQuant 800 image recorder.

Bhattacharya K., Maiti S., Zahoran S., Weidenauer L., Hany D., Wider D., Bernasconi L., Quadroni M., Collart M, & Picard D. (2022). Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life. Nature Communications, 13, 6271.

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