21 Questions 18 Answers 0 Followers
Questions related from Eduardo Alvarez
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...
11 November 2019 230 1 View
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...
07 July 2019 7,374 2 View
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...
06 June 2019 8,397 2 View
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...
04 April 2019 8,854 9 View
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?...
04 April 2019 8,085 4 View
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...
04 April 2019 657 3 View
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...
04 April 2019 8,532 5 View
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...
04 April 2019 1,192 3 View
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...
04 April 2019 3,382 4 View
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...
11 November 2018 7,835 2 View
Good day, is the transfer buffer used for western blot re-usable for other occasions? I use 20% methanol and 10% 1X Tris Glycine SDS (TGS) buffer. I ask because I wish to not waste methanol in...
11 November 2018 9,268 8 View
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...
11 November 2018 4,315 1 View
Good day, I have been having problems with cell counting with THP-1 cells. I counted my cells directly from the flask by resuspending the culture media to promote equal distribution of cells...
08 August 2018 1,694 2 View
I work with suspension cells, specifically, THP-1. I counted the cells before centrifugation ( directly from the flask) and after centrifugation (pellet). Before I had 10 million cells and after I...
08 August 2018 6,716 7 View
Good day, I need to perform a viral load analysis to quantify the number of viral particles/mL found in my samples. The virus that I will be working is with the Human immunodeficiency virus type...
08 August 2018 7,248 3 View
I am recently beginning to understand more or less how to culture suspension cells, but I get confused in regards with passage number in these cells. My question regards to: Thawing: Whenever...
07 July 2018 6,777 5 View
Good day, I have been recommended to expand my suspension cells, specifically, the THP-1 cells by seeding 5 million cells in 100mL RPMI 1640 media and wait for around 1 week and a half until cell...
07 July 2018 6,247 2 View
Good day, I am having problems with culturing my THP-1 cells. I tried culturing them in several different volumes, I was recommended to seed 5,000,000 cells in 100mL to 150mL and let them sit...
07 July 2018 9,776 5 View
Good day, my question is fairly easy and I just want to be very careful for my cultured cells. My cells required 90% FBS and 10% DMSO as freezing media. For this, I would need to add the following...
07 July 2018 9,365 7 View
Good day, I will be receiving a stock of THP-1 cells from a collaboration and I would need to expand the cells. For people who are working with the THP-1 monocytic cell line, at what optimal...
06 June 2018 6,856 2 View
Good day, I am a fairly new researcher having problems in culturing the THP-1 Blue monocytic cell line. I am trying to make a bank of these cells for future use; however, whenever i culture...
05 May 2018 2,589 13 View
