Prestained markers are dissappearing from stain free bioRad gels during the running?

21 December 2020 0 7K Report

Hi! I am having several problems with the running procedure in western blot.

I prepare the acrylamide gels (I use BioRad stain free gels) with fresh APS 10% and TEMED, as I have been always doing. Suddenly one day, while the gel was running HMW of prestained marker began to dissapear, and at the end of the running most of the markers bands had dissapeared too...

after that when I see the bands of my protein in the gel (this BioRad kit allows you to make a photo in Chemidoc to see how gel has ran), all of them has dissappeared...

I have changed these items: running buffer, aps, temed, acrylamide... and the problem remains...

I have read that probably it could be that I have to degass acrylamide solutions (I have never do it and I have never had problems about that), or also the exceed of APS10% could degradates my gel...

Badges
Science topic

More Giovanna Revilla's questions See All

Free software for deconvolution of mixtures in spectroscopic data?

Is there a free software to perform simulations and to deconvolute mixtures of compounds in FORS and Raman data? I would like to: - simulate mixtures of pigments and colorants with FORS spectra -...

15 April 2024 6,393 0 View

Wich RNAlater is better: from Thermo or Sigma?

I'm gonna start to colect biopsy samples from breast cancer and, in order to preserved the RNA for some hours until I can store the sample in the freezer, I'll use some RNAlater. However don't...

29 February 2024 6,147 3 View

Can you suggest literature criticizing the potential offensive implications of the "Neo-tribe" theory (Maffesoli, Cova & Cova, Hardy, etc.)?

Recently, I received peer review feedback that criticized my usage of terms like "neo-tribe" and "neo-tribalism" in a sociological paper. The reviewer found these terms potentially offensive to...

13 August 2023 7,461 0 View

Is there any issue using 12ug/ml of doxycycline for tet/off system?

Hi, I generated a stable cell line with a shRNA. However, when I tested the optimal dose of DOX to silence the target gene I obtained a working concentration at 12ug/mL... So, as the majority of...

07 March 2023 4,725 1 View

Are there any pH indicators emitting in the NIR spectral range?

I'm currently looking for a pH indicator that can be excited in the VIS range (610-630 nm) with emission in NIR range (760-790 nm). I don't have any constrain about the solvent

17 November 2022 6,966 0 View

What is the role and impact of big infrastructures in the extensive and planetary urbanization processes?

I want to understand the way in which large infrastructure works contribute to the urbanization processes of rural areas and natural spaces. This includes its impact in social and environmental...

13 August 2021 3,881 4 View

MrBayes Swap = Lowers ESS?

I am performing mcmc and ss analyzes on MrBayes, with morphological characters, but the ESS are very low. I have already tried to increase the temperature of the swaps and increase the number of...

25 March 2020 4,302 0 View

What is expression of PD-1 on T cell clones during expansion with feeders?

Hi, I would like to know if anyone has expanded CD8 T cell clones with feeders and check PD-1 kinetic of expression. Is it similar to expression on CD8+ cells stimulated with anti-CD3/CD28 beads?...

18 March 2020 6,700 0 View

Where can I access The Greater Cincinnati Well-being Observation Tool?

The Greater Cincinnati Well-being Observation Tool developed by Clarissa Rentz (2005).

15 March 2020 2,728 3 View

What happens to glycerin at 55 °C?

Dear all, Currently, we are trying to produce hydrogen and methane by anaerobic digestion of biodiesel waste (glycerin) at thermophilic temperature (55 °C/ 131 °F). However, as soon as...

10 March 2020 6,805 11 View

Which Scopus Journal provides the most affordable fees?

"PUBLISHING IN A SCOPUS JOURNAL" Researchers are now at a cross road. The critical need to publish in a Scopus or ISI, etc journal is ever vital. Journal Publication fees must be submitted....

10 August 2024 8,621 1 View

Does anyone know what might be causing the smeared bands on my western blot?

I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...

10 August 2024 7,480 3 View

Do I need specific antibodies to stain for a fusion protein?

I am staining some brain sections stored in cryoprotectant that express a Histone H2B- GFP fusion protein that were generated ~10 years ago. I know I need to enhance signal with an anti-GFP...

07 August 2024 5,338 2 View

AUX gas reading problem on QE with full MS and PRM method in one run?

Dear QE-users, In the method where full MS positive mode and PRM mode are used, we always get an incorrect auxiliary gas reading (41 instead of 25). This only happens in this method; other...

06 August 2024 4,953 0 View

How to determine positive-stained cells in FACS? Use isotype or unstained control?

To compare positive and negative cell populations in flow cytometry, should I compare unstained cells with antibody stained cells? Or with the isotype control? Most papers show comparison with...

06 August 2024 6,728 6 View

Why is my ASO degraded quickly on agarose gel?

I have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO...

06 August 2024 3,130 2 View

Can I multiplex a mouse monoclonal and rat primary for IHC (mouse brain tissue)?

Hi all, I was just wondering if anyone has experience with multiplexing a mouse monoclonal primary and a rat primary. I'm trying to multiplex by incubating them in the same well but was told by a...

06 August 2024 9,710 1 View

Low-yield gel extraction problem?

I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...

05 August 2024 9,798 3 View

Could you please suggest methods to compare free protein and immobolized protein binding properties?

I have an antibody binding generic protein and I need to compare its activity in a free and immobolized form. I understand that there are a number of methods to determine Kd value of a free...

05 August 2024 5,311 0 View

People weight in Oaxaca Blinder Decomposition on R?

Hello guys! Do you have experience running a Oaxaca-Blinder decomposition on R applying person weights. How do you suggest doing it? I have a variable PERWT which gives more information on how...

04 August 2024 6,033 0 View