Problems with dose response curves

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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

Optimal cell (HEK-293) density for transient transfection and IgG expression

I am wondering what your opinion is on what cell density is optimal for expression of antibodies by transient transfection of suspension adapted HEK-293 cells in batch culture using PEI as...

01 February 2013 9,611 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

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

Can two EC50 values with different hill slopes be compared?

Hello, I fit a classical dose response curve Y=Bottom + (Top-Bottom)/(1+10^((LogEC-X)*HillSlope)) to my data (not related to inhibitors etc). The model I use is purely qualitative but it fits...

23 February 2021 8,155 5 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

What is the best temperature to coat an ELISA plate?

Hello, I want to control the temperature of my ELISA plate during incubations to get consistent results. In general, what is the best temperature to coat the plate with antibody? I have been...

16 February 2021 2,377 7 View

Is there a way to tell based on appearance which protein crystal should diffract the best?

I have had unheard of success with protein crystallography lately from a super successful protein expression and purification batch. I have attained a lately reproducible vast amount of crystals...

09 February 2021 5,155 5 View

How to measure the concentration of a labeled protein with no tryptophan or tyrosines?

Hey I'm trying to measure the concentration of a protein I've purified and then labeled with Atto 647 flourescent dye. The protein has no tryptophans or tyrosines (although does have...

04 February 2021 4,482 4 View

How to solve error "Cannot find position restraint file restraint.gro (option -r)." on GROMACS?

Hi, I am about to do energy minimization but the above error occurred, I checked my .mdp file and .top file but could not find the problem. TOP file ; Include forcefield parameters #include...

01 February 2021 780 3 View

Why is Fermented Plant Juice (FPJ) always diluted with water? Could it be diluted with other solvents such as rice water?

I am currently making a research paper with my classmates in Philippine Science High School and we are trying to find references that mixed Fermented Plant Juice with other solvents instead of...

17 January 2021 5,099 4 View

Any stable dye to color acrylamide hydrogel beads?

Hi! We are generating hydrogel beads(50-60um) using microfluidics as single-cell beads. To simply all the experiments (especially the centrifuge), I am looking for a dye. The dye should be stable...

14 January 2021 4,383 8 View

How to phosphorylate specific residues on pymol?

Hi everyone, I am using the pyTMs plug in on Pymol to phosphorylate a particular threonine in my protein. I am having difficulty in selecting only one or two residues - it seems I can only...

05 January 2021 230 3 View

Carlos Guillen

You can made it by hand. You can generate the sigmoidal plot and calculate the half of your maximal value of absorption obtained with the ECL. Afterwarsd you can intrerpolate in the graphics to obtained the optimal concentration of your antibody

Anette Martensson

Thank you both of you. I finally managed to do it