Using R&D Quantikine TNFa ELISA on mouse brain lysates?

Buffer to disrupt platelet alpha granules for PF4 quantification?

Hello! I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation. I will use the ELISA from R&D...

03 March 2021 1,499 2 View

Anyone plz suggest relevant topic for research in image processing+Deep learning.?

I have selected brain tumor images ...but now found that already lots of research done n this topic.

03 March 2021 5,774 3 View

Buffer Exchange using column packed with sephadex G-25 effect on protein concentration?

Hello, If i am doing a buffer exchange for an antibody of 1mg/mL, does the elution lose protein in the process of buffer exchange? For example, if i flow through 500 uL of 1mg/mL sample, and...

03 March 2021 6,299 3 View

After a reference from Int J Body Compos Res, can any body help?

Hi, I am after the reference below, my library says it cannot obtain a copy either locally or internationally, any help appreciated! Chris Wang ZM, Heshka S, Wielopolski L, Pi-Sunyer FX, Pierson...

03 March 2021 6,193 1 View

Can we block unwanted images during the x-ray prediction?

i am try to classify the x-ray images. During classification , can i block unwanted images (except x-ray image).

03 March 2021 7,100 1 View

Have someone successfully used the ER alpha (Santa Cruz sc-8002) antibody in immunostaining ?

Santa Cruz now recommends this new antibody (F-10) but I can't find a publication using this reference. I'm working on human et non human primate hypothalamus and so far, it doesn't work. Is...

02 March 2021 10,003 2 View

Sample dilution for quantitative analysis using ELISA?

If the detection range is in ng/ml but the reference range is in ug/ml for a molecule or protein in serum or plasma .how to dilute and what is the initial volume to be taken for quantitative analysis

02 March 2021 7,670 3 View

How optimize the Size Exclusion Chromatography?

Hello! I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and...

01 March 2021 2,215 2 View

Energy minimization in LAMMPS using NVT ?

How do I do energy minimization in lammps with NVT ensemble ? I am using the following command and it does not seem to work. It stops at extremely high energy and gives me the error zero search...

01 March 2021 2,294 3 View

Why are my bands weird in this Agarose gel?

Can someone please give me some possible things that could go wrong? Here is my recipe: 0.5g Agarose 50 mL of TAE 1x 1 uL ethyl bromide. Gel was run at 100V for 1 hour. The buffer used is also TAE.

01 March 2021 9,952 3 View

Maria del Pilar Corena-McLeod

Alberto, RIPA buffer contains about 150 mM NaCl which would be enough to precipitate TNFa since it is a 235 AA or close. Perhaps the salt is precipitating your protein and you are losing it before you load it. I would look into it.

Xiaojie Zhao

Hi Alberto,

I experienced the same issue. I have tried PBS and no ionic-detergent lysis buffer to homogenize the mouse brain, and the ELISA signal still very weak (below 10.9 pg/ml). 600 ug protein I loaded in R&D ELISA KIT, which was pretty much. Please let me know if you get any success in this assay. Thanks.

Xiaojie

Alberto Sepulveda Rodriguez

Maria del Pilar Corena-McLeod Thanks for the advice. I had definitely not thought about that... Do you have any recommendations for lysis buffers that would likely work? I was going to try with homemade PBS-based, and then TRIzol next... But I fear that those two will have the same issues re: precipitation.

Xiaojie Zhao That was going to be my next move, to lyse in PBS + TritonX100 + proteinase/phosphatase inhibitors...

We actually get signals below the detection limit...

I might try a spike+recovery test protocol (pdf attached from R&D) to ensure that the lysate media is indeed the problem.

In parallel, we are are also trying the higher sensitivity version of the ELISA from R&D soon. I refuse to believe that my samples have such low TNFa levels, but maybe with this more sensitive kitI'll be able to detect a relative effect even if I don't trust my absolute values.

Alberto,

Spike testing is a good idea. let me try. Also western blot and immunohistochemistry methods are the candidates, but ELISA owns the priority. I will let you know my results.