How to image cells on inserts with Cytation 5?

Having trouble detecting phosphorylated HSL. Any tips for how to prevent de-phosphorylation events or detect phosphorylation?

I face many problems like:

1. Weak bands.

2. not straight bands.

3. some times I got no bands.

Hello everyone! I'm currently having some trouble looking for software that can view and quantify Western blot bands. Does anyone have any recommendations? I am currently using a MacBook Pro for my research.

*Before you comment with any of the generic troubleshooting advice from the front page of Google, just know I've already tried all that*

No matter how I alter my conditions for wet tank transfer, there is still always A LOT of protein left in my gels. After transfer I always check my membranes with Ponceau S, and there are full lanes of protein on the membrane itself, but coomassie staining of the gel shows a lot if left behind. So my trasnfers are incomplete.

Our lab does a wet tank transfer using the BioRad mini protean boxes with PVDF membranes and Towbins buffer (20% methanol) for 300mA for 1-2hrs (depending on target protein size). So far they all seem to be coming out like the attached picture. I have tried using constant voltage ranging from 60-100V, constant amps ranging from 300-400mA, increasing transfer time for up to 3hrs. I've tried replacing every single buffer and reagent used in the entire western process from gel cast through transfer. I've used no methanol and up to 30% methanol. Tried using different boxes, power units, and electrical outlets. Tried everything from 5% gels to 15% gels and gradients all at different thicknesses (.75-1.5mm), and they all come out more or less the same.

The only thing that helped a little was adding SDS to the Towbins buffer, which still left SOME protein in the gel. But not nearly as much. However that resulted in the lower weight proteins blowing through the membrane. I tried contacting BioRads western blot tech service but all they did was suggest the same generic advice for troubleshooting.

Has anyone experienced this? I am waiting on commercial gels to come to as a last ditch effort to see if some how one of the components used in our gel casting we bad but other than that I'm out of ideas for solving this.

List of steps used or troubleshoots tried:

Tissue/cells homogenized in RIPA

Samples are prepped by boiling in laemelli buffer + BME for 10mins

Gels are cast fresh the day of

Gels and sandwich components are soaked in transfer buffer for 15mins prior to transfer.

Transfer is done on ice with a cooling pack.

PVDF membranes are used and activated with methanol prior to use.

All buffers are made fresh.

10-40ug of protein are typically used per lane and all ponceau staining after transfer shows an OK amount of protein of all weights make it to the membrane, just a lot is left in the gel.

Varying amounts of methanol and SDS have been used in transfer buffer.

Varying transfer conditions and transfer boxes have been tried.

pH of all buffers and even DI water have been checked.

Hi, everyone

I was reading many PCR protocols where I found that 100 pmol = 100uM. I am unable to understand this conversion as I found in google that 1pmol = 1000000 micromol.

can anyone guide me in a detailed way?

Thank you

Hi everyone,

I am culturing Jurkat T cells E6.1 obtained from ATCC. This cell line is known to be in suspension forms. The cells were expanded as as suspensions for the first few days after reviving but then became adherent. The culture media used for culturing is made by ATCC protocols (RPMI + 10% FBS). Please let me know if anyone also experienced this and if there are any suggested treatment. Thank you for the help!

Hi All,

I have heard that papers in Nature, the ones that got into review and further consideration are often held for a year and more in limbo. Do other people have the same experience and what action should be taken? Obviously you want your research to go out and at some point publishing in another journal may be a better option.

The new service is ResearchSquare teamed with Nature now allows you to post a preprint and get a doc number, same is for Archive. Do people have experience with posting preprint online and if this would violate Nature embargo policy?

What is the difference between melting temperature and annealing temperature?