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

How to perform HIV RNA isolation and quantitation?

Good day, I have been able to prepare stocks containing HIV-1 pure, frozen in liquid nitrogen in cryovial tubes. For the purpose of my research, I need to quantify the virus to know how much...

10 November 2018 7,768 2 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

No title

02 March 2021 3,060 3 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

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

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

No title

28 February 2021 9,936 2 View

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 3 View