HPLC - Chemstation calibration curve?

Hi everyone, I know I'm asking a lot but I'm learning HPLC for the first time. I'm working with a simple assay containing three simple compounds that separate well. I've recently inject standards...

02 March 2019 3,091 4 View

Toxic protein causing cell lysis?

Hi everyone, I'm currently conducting protein expression trials with a difficult to express enzyme. I'm trying a range of induction times, temperatures and induction OD. I notice that at a lower...

02 March 2019 327 5 View

Improving sequencing results?

Hi everyone, I'm sequencing a gene post site directed mutagenesis and so far I'm getting horrible results with poor coverage. For the same gene I sequence twice using the T7F and T7R primers. I...

05 June 2018 8,908 3 View

Tips for getting gene mutants sequenced?

Hi everyone, I'm trying some site directed mutagenesis at the moment and having problems with the sequencing. I'm new to sequencing so I thought I'd try to get some tips. I'm currently using...

31 December 2017 700 2 View

Can anyone recommend a good protocol for site directed mutagenesis or should I use a kit?

Hi guys I'm looking to use SDM to change a single residue in an enzyme I'm working with. I'm new to this so I thought I would ask advice. Is there a current protocol that works best or should i...

10 November 2017 625 5 View

Question about stability of PNP substrates in kinetics assays?

Hey guys I'm working with PNP-acetate and PNP-butyrate, two model substrates for esterase/lipase reactions which yield a product which absorbs at 405 nm on hydrolysis. All of the assays I've...

09 October 2017 9,094 5 View

Protein migrating at lower molecular weight, proteolysis or wrong protein?

Hey guys, I've recently cloned a protein into a vector and attempted expression. Colony PCR and gels confirmed an insert of an appropriate size was in the colony that I selected for protein...

05 June 2017 162 5 View

Removing contamination from DNA

Hey guys I've been using the Qiagen gel extraction kit to purify my DNA post restriction digest. I seem get very low yield but my big problem is the purity of the DNA product. When I spec it I get...

05 June 2017 1,461 4 View

Can I use CRISPR to knock out a gene in mycobacterium?

02 March 2017 1,193 25 View

Should Researchgate have a contributor of the year?

I think there should be. We could have one for each category. So researchers put in a considerable amount of time to helping people with their research. What does everyone else...

02 March 2017 530 0 View

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

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

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

What do I do with DLS peaks that are from the buffer?

I'm looking at the aggregation of my protein sample using DLS. Unfortunately, my buffer (20mM HEPES) also results in a set of peaks. These are at approximately 1 and 1000 d.nm. The lowest peak...

01 March 2021 9,015 2 View

Why initial concentration of Pb not the same as calculated one when testing using AAS?

I am currently doing research on the adsorption of heavy metal using magnetic reduce graphene oxide. I use AAS to determine the concentration of the heavy metal. I prepare the Pb solution with...

26 February 2021 9,580 4 View

Is there anyone who experienced with cytotoxic effect of Hoescht 33342 dye?

Is Hoechst toxic to cells? In general, it is known as non-toxic. I am working with dsRED MDA-MB-231 cell line. I applied it for my PDMS-based microfluidic platform, for this reason, I incubated...

26 February 2021 9,119 3 View

What is the most suitable resuspension buffer for TCA/acetone precipitated protein?

I need to extract protein from fermented carbon sources for Bradford assay. Most researchers experiencing insolubility of pellet in resuspension buffer. Please assist me to select most suitable...

26 February 2021 7,129 3 View

What urea buffer can i use for biotagged protein extraction from streptavidin beads?

Hi all, I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by...

24 February 2021 5,749 3 View

Cloudy/high background western blot - can anyone help me?

Hi everyone! I try to detect VDR protein (~48 kDa) using Western Blot. After SDS-PAGE using 10% MP TGX Stain-Free Gel from BioRad I transferred the protein onto an metOH activated PVDF membrane...

23 February 2021 6,266 3 View