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Questions related from Kristen Navarro
Hello! I am planning a western blot experiment, and I am looking for GAPDH antibodies to order. In the past, I have experienced a lot of hit-or-miss when ordering antibodies based on just the...
08 July 2024 1,631 0 View
Which membrane material is the best for capturing and visualizing phosphorylated proteins in a western blot? Or is there no difference?
06 June 2024 9,385 2 View
Hello everyone! My PI would like me to measure the amount of mitochondrial DNA in multiple cell lines using qPCR targeting COXI and COXIV. However, I have never done a qPCR that measures...
04 June 2024 6,741 1 View
Hello! I am trying to analyze some RNAseq data I recently received. I would like to craft a heatmap comparing the expression of specific genes across my cell samples, but I am not sure which of...
09 May 2024 5,620 0 View
Hello everyone. I am in the middle of performing western blotting experiments measuring expression of p-S6(Ser235/236) protein in total cell lysate. Originally, I was quantifying my data using...
12 April 2024 4,435 1 View
Hello! I need to snap freeze cancer pellets to submit for multiomics. Does anyone know of 15 mL conical tubes or Eppendorf/microcentrifuge tubes that can withstand liquid nitrogen and dry ice?
16 February 2024 4,178 1 View
Hello! I am not sure whether or not my protein samples are reduced or not. Based on the information below, can someone please let me know if they are reduced? Sample setup: __ uL total cell...
13 February 2024 4,885 1 View
Hello all! I need to make 100 mL of the following solution. I tried to use the C1V1 formula, but I have dyscalculia so my math skills are non-existent. Ponceau-stained membrane can be detained...
02 February 2024 8,933 1 View
Hello everyone! I'm currently running an in vitro experiment where I test the effects of treating human cancer cells with "starvation media" (aka DMEM media with no FBS) on mTORC1 signaling. I...
19 January 2024 4,341 1 View
Good morning. I recently performed a BCA, and I obtained the following values for my protein concentration. I formula I used in Excel was: =(E39-0.0798)/0.0377 I then multiplied the value by 10 or...
02 January 2024 3,143 3 View
I am brand-new to the world of protein research. I have total cell lysate samples that I would like to extract RAB5 and RAB7 proteins from. Which is the best approach to do this?
17 October 2023 178 1 View
For my experiment, I would like to measure phosphorylation of RAB5 and RAB7 in human cell lines by using a kinetic assay. Is there a kit that would be able to do this?
16 October 2023 4,790 0 View
I need to design qPCR primers for my experiment, but PerlPrimer (a software I used in the past) is not easily downloadable on Macs. For my qPCR primers I need the following attributes: Annealing...
19 September 2023 4,011 3 View
Hello everyone! I need to perform a MTT cell viability assay, but I am not sure which kit would work best with our lab's plate reader. Below is an image of the absorbances that our machine reads:
08 September 2023 6,884 1 View
Hello! I am trying to design sgRNAs for dCas9 fluorescent tagging, but all of the tools I find online are for standard CRISPR knocking in/out. Anyone have a recommended tool for dCas9?
28 August 2023 1,922 0 View
Our lab usually uses RIPA buffer, but mostly on total cell lysate and mouse tissues. I want to isolate proteins in cell culture media, but I am not sure if using RIPA buffer is ok? I also saw...
24 August 2023 1,884 4 View
Hello everyone! I am reading a protocol for CRISPR using a dCas9, and I came across the following passage in the gRNA section: A. Design sgRNA oligos Fwd (all): ACCGNNNNNNNNNNNNNNNNNNN Rev (Sp):...
21 August 2023 9,080 0 View
Specifically, one of human cell lines?
21 August 2023 5,780 6 View
I am trying to find a decent protocol, but the only ones I see are for generating nuclear, mitochondrial, and cytoplasmic fractions.
14 August 2023 3,159 1 View
Hello! I am performing a lentiviral transduction on human fibroblasts for the very first time. The vector I am trying to insert contains puromycin and ampicillin resistance. How much ampicillin...
07 August 2023 5,487 4 View
Hello! I need to perform a DNA gel extraction, but I do not own an open UV lamp or lightbox to do it. I need suggestions/help for finding the best options. Does anyone have any recommendations?
31 July 2023 7,912 1 View
Hello everyone! I am trying to generate melting point curves for my qPCR samples, but my overall amplification is failing. For some reason, my amplification is starting later than desired. (I am...
20 July 2023 3,230 0 View
Hi! I am trying to optimize a qPCR experiment using mouse tissues. I am trying to use a GAPDH control, but I think my primers may not have been designed well. Does anyone have primers for GAPDH...
18 July 2023 5,896 3 View
Hi everyone! I am trying to find the DNA sequence upstream of the endogenous COPB1 gene. (ENSG00000129083) However, I am having a hard time finding it, even in the genome viewer. How do I find the...
13 July 2023 6,563 3 View
Hello! I am trying to determine the best controls for my western experiment. I was wondering what the best controls are with a small kDa size?
07 July 2023 577 3 View
I have never done a co-IP experiment before, so I have no idea what to use as my negative and positive control. For my sample, I am using human fibroblast total cell lysate.
29 June 2023 988 1 View
I recently ordered a cloning product from Thermo Fischer, and the instructions for primer design require that the forward primer have the Kozak sequence ([G/A]NNATGG) on the 5' end. However, I...
26 June 2023 5,579 1 View
Hello! I am trying to create a primary antibody that targets a specific region of my protein of interest. What is the best software/web tool for protein antibody design?
16 June 2023 2,522 0 View
Hello! I need help making a 25 mM HEPES buffer with a pH of 7.4. I've tried calculating the dilution, but I keep messing up. The stock solution I am planning to use is: 30 mM HEPES Buffered...
05 June 2023 7,778 1 View
My graduate work was in C. elegans, and this is my very first time performing live cell imaging in human cell lines. (fibroblasts) I would like to use live cell imaging to track two proteins of...
19 May 2023 1,090 2 View
Hello everyone! I'm currently having some trouble looking for software that can view and quantify Western blot bands. Does anyone have any recommendations? I am currently using a MacBook Pro for...
24 March 2023 670 4 View
Hello! I am trying to find a plasmid and protocol to overexpress a specific gene of interest in fibroblasts. Does anyone have any recommendations?
24 March 2023 2,864 0 View
I recently ran a Western blot, and some of the bands appeared to be partially missing, as if they were cut in half. I looked online for possible solutions, but most troubleshooting guides do not...
10 March 2023 679 2 View
Hello everyone! I am trying to find a protocol to isolate microglia from mouse embryos. (the mouse model I use produces homozygous lethal pups) All of the protocols I have read are for adult mouse...
14 February 2023 8,006 1 View
Hello! The sequences I am examining in the Ensembl Database are in cDNA format only, but I need to look at the RNA sequence. How can I convert the cDNA sequence into RNA? Most online tools only do...
09 February 2023 1,194 3 View
I'm trying to use EzMol to illustrate a protein sequence, but there are no PDB files for the sequence I'm trying to analyze. (NCBI: XP_006501616.1) How can I create a PDB file?
08 February 2023 7,817 3 View
