18 July 2023 0 6K Report

Greetings fellow researchers.

I am doing experimental work on normal human epidermal keratinocytes, adult (NHEK) monolayer cell culture and I am interested to analyse the proliferation and differentiation markers (keratin filaments such as keratin 16, K16 and involucrin) using sandwich ELISA with my test material in inducing acne vulgaris mechanisms. There are several questions that I am in doubt:

  • As we all know that these keratin filaments are located intracellularly, is it more appropriate for me to use cell lysate as my sample in ELISA measurements?
  • If that is the case, can I use commercial RIPA lysis buffer (with protease inhibitor addition) to lyse the keratinocytes and get these keratin filaments?
  • Are these keratin filaments ok to be dissolved in the RIPA buffer and directly act as samples in my ELISA measurements, and are they ok to be stored at -20 or -80oC for my later measurements?
  • Do I need to do total protein quantification assay, for example bicinchoninic acid assay (BCA) for my cell lysate in order to normalise the concentration of my proteins of interest (K16, involucrin) measured from ELISA with the total protein concentration for my data analysis later? Since the RIPA buffer is expected to release all proteins from the cells as well.

    • I appreciate your kind response and advice on this. Thanks!

    More Ming Xian Ang's questions See All
    Why am I unable to coat oleate-capped NaCeF4:Gd,Tb nanoparticles with mesoporous silica shell?

    My oleate-capped NaCeF4:Gd,Tb NPs have been synthesized via the thermal decomposition method. 1mmol of NPs has been dispersed in 20 ml of cyclohexane. I have tried dissolving 1 ml of NP in...

    01 December 2020 4,713 2 View

    How to explain reason for using 0.1 as margin of error?

    I currently conduct research on factors that influence career choice and I decided to use 0.1 as the margin of error. My supervisor asks me to explain the reason the value is selected. What reason...

    23 November 2020 4,764 2 View

    How to analyze correlation of multiple answer dependent variable with independent variable using SPSS?

    My research had a multiple answer dependent variables. How can I use spss to find the correlation of the dependent variable and independent variables? Should I use define multiple response set first?

    23 November 2020 753 3 View

    Machine Learning and Big Data Analytics Tools Question?

    What would be the best big data analytical tools to determine changes in behavioral health (e.g., addiction behaviors)? The data used to predict behavioral health changes are secondary data which...

    30 September 2020 9,522 8 View

    How is the quality of archived blood sample for HbA1c test?

    HbA1c is an important indicator of long-term glycemic control with the ability to reflect the cumulative glycemic history of the preceding two to three months. Also, HbA1c is measured as the ratio...

    16 September 2020 2,423 4 View

    How to gain the access to image recognition?

    I am graduate from other major, and now working in computer science and technology, my roomates are mostly researching on image recognition. I want to switch to it and learn how to cooperate with...

    21 August 2020 2,888 9 View

    What are the current research results on est- SSR primers in azaleas?

    I plan to conduct research on est-ssr primers of rhododendrons. I would like to know the research results that have been completed so far. If anyone can give me some Suggestions, I will be very...

    31 May 2020 3,936 3 View

    What is a good rice variety to research on red, brown, black and white rice towards antioxidant properties and anti-alphaglucosidase on diabetes?

    We will be conducting research on colored rice--red, brown, black and white rice and utilize FRAP, superoxide assay and hydroxyl scavenging assay to determine antioxidant activity and correlate it...

    20 April 2020 7,334 3 View

    Is there a catalogue of Trichoderma isolates? How are these isolates given names?

    Hello everyone, I've been trying to find out how certain isolates are given a name and where to find a list of established names. e.g. there's T. asperellum T54, T. spirale T4, T. hazarnium T40...

    10 April 2020 3,862 3 View

    Why did I get so many negative clone after electronic shock when constructing a phage library?

    I use phagemid pCDisplay3 and M13KO7 to construct Human Naive phage library. before digested with SfiI , I prepared ScFv (750bp) using gel purification. After electronic shock , ,plating on...

    26 March 2020 4,023 2 View

    Similar questions and discussions
    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

    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

    No title

    02 March 2021 3,060 3 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

    Can NFL theorem be valid in infinite search space in ML?

    NFL theorem is valid for algorithms training in fixed training set. However, the general characteristic of algorithms in expanded or open dataset has not been proved yet. Could you show your...

    01 March 2021 1,189 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

    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

    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

    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

    Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

    I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

    01 March 2021 3,607 3 View

    Our partners will collect data and use cookies for ad personalization and measurement. Learn how we and our ad partner Google, collect and use data. Agree & close