How to perform HIV RNA isolation and quantitation?

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Why am I getting smears on my western blot bands?

Recently, I have had problems with western blotting where I get smears all across the lanes. I used to get clean bands at some point but now it is all smears. Is this due to insufficient washing...

10 November 2019 107 1 View

How can you stain unstained ladders PRIOR running gel electrophoresis for western blots?

Good day, I have seen that our laboratory has a lot of stock of Precision plus unstained standards and very few of Precision plus stained standards. Since I need to run a lot of western blots in...

06 July 2019 7,301 2 View

Why does the secondary antibody blue light signal fade so quickly?

I have been doing some troubleshooting relating to western blot with the help of a friend. We decided to test if our anti-rabbit conjugated HRP secondary antibodies are working properly by...

05 June 2019 8,324 2 View

Why am I having so much background in my western blots?

I have been trying to look for my protein of interest (phosphorylated PKR) in my western blots and I have been having trouble because there appears to be a lot of backgrounds. Sometimes, I can see...

03 April 2019 3,323 4 View

What blocking buffer should I use for antibodies from Santa Cruz?

Hello, I have been having a difficult time with western blot lately and I was wondering, what blocking buffer and antibody buffer should I use when using antibodies of Santa Cruz Biotechnologies?...

03 April 2019 7,994 4 View

Can Laemmli buffer buffer be re-used for future samples?

Good day, I prepare Laemmli buffer 2X from Bio-Rad by adding 950uL of Laemmli buffer + 50uL of B-mercaptoethanol for a final volume of 1mL for SDS-PAGE. Since a lot of Laemmli buffer still...

03 April 2019 8,445 5 View

Bands cannot be seen with Ponceau S staining? Should I continue with western blot?

Before running the SDS-PAGE, I added pre-stained Kaleidoscope on the well corresponding to the ladder, 30ug of proteins from my cell lysate samples and then as another marker, I added again...

03 April 2019 8,779 9 View

Can I re-use phospho antibodies after blocking membrane with milk?

I recently performed western blot and blocked my PVDF membrane with 5% non-fat milk. After blocking, I did 3 washes with 1X TBST for around 5 minutes each. Then I diluted my primary monoclonal...

03 April 2019 551 3 View

Western blot problems. Why can't the protein bands cannot be seen?

I have been running into some difficulties with western blotting. I never see any protein bands in my western blots. I run my western blots in wet conditions and with PVDF membrane. I activate the...

03 April 2019 1,115 3 View

What are the parameters for the transfer of western blot?

Good day, I recently did western blot up until the transfer part, However, the proteins were not transfer from the gel to the PVDF membrane. The staining of Laemli buffer and Kaleidoscope were not...

10 November 2018 4,251 1 View

What is your perception on using visual analysis or quantitative approaches to evaluate data from single-case research designs (n of 1 trials)?

Results of single-case research designs (i.e., n-of-1 trials) are often evaluated by visually inspecting the time-series graph and computing quantitative indices. A question our research team is...

03 March 2021 687 1 View

In PRIMER, is it possible to downweight certain taxa in multivariate community datasets to reflect uncertainty around identification??

I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...

03 March 2021 8,066 4 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

How to quantitative analize the data of TNBS assay for gelatin?

Estemeed colleagues, I found some issues regarding the quantification of the data for TNBS assay. There are different protocols on how to perform that but it is clear to me the "fil rouge" that...

02 March 2021 2,616 1 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

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

Do you know good and user-friendly programs to measure plant quantitative traits?

For my research i will need to measure plant quantitative traits (especially leaves area and roots length, but would be nice to add some more information). I recently discovered...

01 March 2021 5,035 2 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to calculate the annealing temperature of a primer pair?

I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...

01 March 2021 360 7 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View