What concentration do you use to elute a NiNTA resin?

The protocol of Thermo Scientific NiNTA resin (cat. 88221) for purification of proteins containing a hexahistidine tag use an elution buffer containing 250 mM imidazole. For fun after this elution...

07 August 2019 6,108 6 View

Dehydrogenase enzyme activity: would you take a look at my data?

My experiment involve a dehydrogenase and I would like to identify the optimal substrate. For this an assay was established based on the use of DCPIP as cofactor, which when reduced the absorbance...

31 December 2018 9,454 7 View

Potassium cyanide in redox enzyme characterization?

My goal is to characterize a dehydrogenase, we are unsure of which cofactor it can uses but we tried DCPIP as artificial redox cofactor and there is no activity. I would like to try phenazine...

05 June 2018 7,187 4 View

Steady state kinetics: time frame between replicates must be the same?

I am measuring dehydrogenases enzyme kinetic parameters via Michaelis Mendel fit, the enzyme activity is determined by the inverse of the slope in the absorbance of a redox indicator when this...

04 May 2018 6,955 0 View

Methods to stop enzyme reactions?

I checked the previous answers :) So my enzyme kinetic assay is based on DCPIP absorbance at 3 mM, which is outside of the linear optical range to measure continuously on a plate reader. Then I...

03 April 2018 8,655 9 View

Why NIST charges for their spectral libraries?

NIST, the National Institute of Standards and Technology publishes reference libraries of hundred of thousand of compounds. I would like to use these in my computer for GC data, however it looks...

03 April 2018 6,523 3 View

What is the CKBB buffer?

In Gao, et al (NAD-Independent L-Lactate Dehydrogenase Required for L-Lactate Utilization in Pseudomonas stutzeri A1501, doi:10.1128/JB.00017-15) CKBB buffer is employed for the optimum pH and...

31 December 2017 3,938 3 View

How high can I go with maltose on a MBP purification?

I have a maltose binding fusion protein being purified with the MBPTrap column (GE). I get very little protein (less than 1 mg from a 400 mL E. coli BL21 (DE3) culture) during the elution step...

10 November 2017 5,667 8 View

FPLC default wavelenghts: what can you measure at 215, 255 and 495 nm?

The default settings of the FPLC UV detector are for wavelengths 215, 255, 280 and 495 nm. I know that the 280 nm is the most important since it directly measures protein via the aromatic ring in...

07 August 2017 9,234 4 View

How can I obtain legible GI sequences?

02 March 2015 6,978 4 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

Waters 2707 HPLC autosampler problem?

I am using a 2707 waters HPLC device. When I try to inject a sample, it says missing plate or rack. I changed the needle and calibrated its position but I still get the same problem. I even get...

02 March 2021 1,408 1 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

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 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

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

Any research papers for extraction of nutrient deprivation on macroalgae? Analysed by HPLC-UV and GC/MS. Possibly from macroalgae genus Rhizoclonium?

I am doing my thesis on the nutrient deprivation on the macroalgae Rhizoclonium sp. I will be analysing the levels over a period of roughly 5 days by HPLC-UV and GC/MS. I will be using seawater as...

28 February 2021 6,477 1 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View