Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

Heda, Ghanshyam D.; Omotola, Oluwabukola B; Avery, J.

doi:10.7171/jbt.16-2703-004

Cited by 3 publications

(4 citation statements)

References 18 publications

(15 reference statements)

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“…PVDF membranes are generally recommended for resolving high-molecular-weight proteins. However, in our current and previous studies [34], supported NC membranes produced stronger signals for CFTR, a relatively high-molecular-weight protein.…”

Section: Effects Of Membranes and Blocking Agents On Western Blottingcontrasting

confidence: 59%

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Optimization of Western Blotting for the Detection of Proteins of Different Molecular Weight

et al. 2020

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Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0–20%) in Towbin’s transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins.

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Section: Effects Of Membranes and Blocking Agents On Western Blottingcontrasting

confidence: 59%

“…Band intensities were quantified by Quantity One R 1D analysis software (Bio-Rad) as previously described [34]. The density volume within each protein band was measured as intensity/mm 2 .…”

Section: Discussionmentioning

confidence: 99%

Optimization of Western Blotting for the Detection of Proteins of Different Molecular Weight

et al. 2020

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“…Analysis of protein molecules can be carried out using the SDS-PAGE method. This method was used to detect predicted AtRKD4 protein bands with a molecular weight of ~28.53 kDa by electrophoresis separation (Heda et al 2016). The isolated total protein from leaves of transformant plant carrying the construct of 35S::GR/UAS::AtRKD4 after 5 days treated by 15 μM DEX, and 3 mgL -1 TDZ showed 7 protein bands with various molecular weight.…”

Section: Induction and Detection Of Atrkd4 Protein In Orchid Transformant Plants After Being Induced By Using Dex And Tdzmentioning

confidence: 99%

Detection of AtRKD4 Protein During Induction of Somatic Embryogenesis in Dendrobium lineale Rolfe Transgenic Orchids Carrying 35S::GR::AtRKD4

Wirajagat

Febryanti

Puspitasari

et al. 2021

J.Tropical Biodiversity Biotechnology

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Dendrobium lineale is an Indonesian native orchid from the Spatulata section in Orchidaceae Family. This orchid is important because it is usually used as a parental plant in orchid breeding and is predicted to have a potential phytochemistry compound. In addition, in their natural habitat, this orchid is threatened due to forest exploitation and natural disaster. Therefore the precision mass propagation techniques for this orchid need to be conducted. Biotechnological approaches through inserting embryo gene such as AtRKD4 from Arabidopsis thaliana has already been successfully conducted. This study aims to check the integration stability of T-DNA harboring 35S::GR::AtRKD4 from ten selection transformants and to detect the existence of AtRKD4 protein after induction by Dexamethasone and/ Thidiazuron. The result showed that T-DNA were stably integrated into the genome of D. lineale transformants and the AtRKD4 protein with a molecular weight of 28.53 kDa was detected in D. lineale transformant plants after being induced by 15 µM DEX and 3 mgL-1 TDZ for 5 days.

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“…Protein lysates were prepared and subjected to standard SDS‐PAGE and Western blot analysis, as described . For primary and secondary antibody were used BMP21‐C and 2040–05 (southern biotech) antibodies, respectively.…”

Section: Rt‐pcrmentioning

confidence: 99%

Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis

Bayat

Shojaei²,

Bahrami

et al. 2017

J Biomedical Materials Res

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The aim of the present study was to assess the recombinant bonemorphogenetic protein 2 (RHBMP-2) with higher substantively and solubility for use in calcium phosphate scaffolds for better release in differentiation of mesenchymal stem cells to osteoblast cells. Using bioinformatics tools, two mutations (p. L10D and p. S12E) were chosen and applied in BMP2 CDS sequence to increase interaction with calcium derived composite. The new recombinant mutated sequence (BMP2 ) was synthesized and then subcloned to expression vector pBV220. Experimental data regarded functional protein expression in E. coli. Since no modification was made in the active sites of proteins namely β-sheets and α-helixes, not only was there any change in the specific activity occurred in the specific activity of the enzyme in comparison to its commercial counterpart, but also mesenchymal osteogenesis occurred more efficient on biphasic CaP scaffold model. As we hypothesized, use of negatively charged amino acids such as aspartate and glutamate in protein loops increased the interactions of BMP2-Ca and resulted in its slower and more sustained released from CaP scaffolds compare to commercial RHBMP2. Our data suggested that new BMP2 have greater osteoinductive capacity than RHBMP2 in the same time and dose than RHBMP2. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2799-2805, 2017.

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Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

Cited by 3 publications

References 18 publications

Optimization of Western Blotting for the Detection of Proteins of Different Molecular Weight

Optimization of Western Blotting for the Detection of Proteins of Different Molecular Weight

Detection of <i>AtRKD4</i> Protein During Induction of Somatic Embryogenesis in <i>Dendrobium lineale</i> Rolfe Transgenic Orchids Carrying <i>35S::GR::AtRKD4</i>

Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis

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