Why stop reaction between HRP and TMB to read absorbance at 650nm?

I am working with ELISA and use HRP conjugate as detection antibody and TMB substrate. All of the protocols I have used so far the TMB substrate reaction has been stopped by adding an acidic...

11 December 2018 6,538 4 View

Why does my standard curve not generate expected concentrations in the back calculation?

I am setting up a direct ELISA and have a standard curve which I fit to a 5-PL curve. When I try to calculate the concentrations of the points in the standard curve using the obtained 5-PL curve I...

02 March 2018 1,222 3 View

Is it possible to efficiently conduct E coli cell lysis for protein purification with ultrasound bath?

I want to lyze my E. coli for protein purification but I only have an ultrasound bath, not a sonicator with a probe. Does anyone have any experience of this? Any tips or protocols that can help me.

11 December 2014 1,780 9 View

Can anyone help with "Shadows" of bands on non-reducing a SDS gel?

I have run a non-reducing gel with samples of a few antibodies. The samples are supposed to be pure, i.e. only containing the antibody of interest. I observe some bands below the main band. Does...

07 August 2014 9,815 7 View

What is a good software for curve fitting?

I am looking for a good software for fitting curves and would like to hear what software other people recommend. I want to be able to fit 4- and 5- parameter logistic curves and linear regression....

03 April 2014 6,133 45 View

Difference between FreeStyle 293 (Gibco) and Pro293-DM (Lonza)?

I am fairly new to cell cultivation and I am wondering about the main differences between the FreeStyle 293 media from Gibco and the Pro293-DM from Lonza. As I understand it both are serum and...

04 May 2013 9,474 2 View

How to conjugate dGDP to protein?

I want to conjugate cyclic GDP to a protein to use in immunization for antibody production. Can anyone help me with a way in which I can do this conjugation?

31 December 2012 7,298 10 View

Why fix cells before ELISA?

I have a basic question I wish I knew the answer to. In several protocols for doing ELISA to study internalization of receptors or internalization of antibodies I see that they fix the cells...

11 December 2012 7,417 6 View

Protein concentration using Amicon Ultra spin column, adsorption on membrane?

I am trying to concentrate a protein using Amicon Ultra centrifugal device. I have done this several times before without any problems. This time I had a larger volume of my sample so I had to...

09 October 2012 7,488 34 View

Protocol for internalization of antibody?

I want to set up an assay to detect internalization of an antibody in HEK cells. I want the assay to be ELISA based since I don't have access to a FACS machine or a confocal microscope. Does...

09 October 2012 7,935 21 View

Can we classify HV and LV lines based on line length?

I have dataset which shows the length of power lines. I need to classify the lines based on the line length. Is there a rule to classify the High voltage (HV) and low voltage (LV) lines based on...

03 March 2021 4,116 4 View

Why does blocking with my immunogenic peptide cause nuclear fluorescence in ICC?

Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the...

03 March 2021 5,999 3 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

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02 March 2021 3,060 3 View

Is There Any Feasible Method To Test Fluorescent Compounds Other Than Fluorescence Spectrometers ??

Is There Any Feasible Method To Test The Efficiency Of Fluorescent Compounds Other Than UV Spectrometers ? Suggestions Would Be Appreciated !

02 March 2021 5,785 3 View

Does Napabucasin soluble in dichloromethane and in ethanol?

I need to prepare a solution of Napabucasin (inhibitor of cancer cell stemness) in dichloromethane. The drug is expensive, so maybe somebody already tried to do it. If it is soluble, what is the...

02 March 2021 9,945 3 View

Help request in CST microwave?

im working now on TPV solar cells and metamaterials and i realy need our help in CST microwave i have a tpv cell simulation and i want to extract particullarly the absorbtion of only the active...

02 March 2021 2,246 2 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View