What volume can I apply on desalting column?

06 June 2012 0 7K Report

I have read that for SEC the volume of sample should not be more than 1-2%.

But now I'm doing desalting on Sephadex G-25 (3x50cm column, dead volume is ~180 ml). I guess I can apply more 2%, because I want to separate only two peaks, so technically, I could apply about 150 ml, right? Because the proteins will elute earlier (150. min) than salt will start (180. min). Or are my thoughts wrong?

Badges
Science method

More Tomáš Hluska's questions See All

Is there multiple-alignment tool which would not neccassarily align all sequences together?

When I get results from sequencing, they often overlap only partly or not at all. In such case it's easier to align them with the "parental" sequence (of what I expect), to see where they align....

01 February 2020 8,336 3 View

A simple method for quantitative separation of apoplast and plant cells?

Hello everybody, I wish to separate apoplast from the rest of the plant (i.e. intact cells) to see whether the compound I treat the plants with goes inside or stays outside. When I mentioned this...

07 August 2019 2,886 3 View

How to make a dot plot?

How do I make a dot plot like in this paper Show the data, don't conceal them in Figure 1? All I'm finding on the Internet are only this type of dot plots:...

05 June 2019 2,092 5 View

How can I improve binding to chitin beads?

I express my protein in pTYB12 vector as fusion with intein and then try to purify on chitin beads. Typically, I load overnight at low flowrates (something like 0.3 ml/min). The loading buffer is...

04 May 2019 2,589 7 View

How to determine mode of action of a certain compound?

I'm working with plant hormones cytokinins - derivatives of adenine with a side chain at the N6-group. They are mostly active as free bases (with no modification on the adenine core), or maximally...

02 March 2019 3,322 11 View

I have two questions regarding adenine conjugates at N9 position /?

My first question concerns lupinic acid - conjugate of adenine derivative with alanine https://commons.wikimedia.org/wiki/File:Lupinic_acid.svg The question: What type of bond is it between the...

08 September 2018 8,949 0 View

Is the cell lysis efficiency by ultrasound affected by cell density and/or the volume?

Hi everybody, I'm currently expressing proteins in and extracting from bacteria. The procedure is that I pellet the bacteria from bioreactor, resuspend in buffer (50 mM Tris/HCl, pH 8.0; 1M NaCl;...

07 August 2018 2,453 9 View

I have a few questions about growing E. coli in bioreactor - and the obligatory question mark?

Hello everybody, I'm currently growing E. coli in bioreactor and I have experience only with P. pastoris in bioreactor, so I'm not sure, how should it behave. With Pichia, one can try hard to...

02 March 2018 9,364 4 View

Does ligand have affinity towards protein or vice versa?

In the texts, it's often written, that the ligand has affinity towards a protein (with appropriate affinity constant). However, to me it always occurred that the protein binds the ligand and...

09 October 2017 6,994 0 View

How common is substitution at position 7 of adenine ring?

Some old papers (from the 70s) claim, that the substitution at position 7 is rare in the nature https://www.ncbi.nlm.nih.gov/pubmed/486500https://www.ncbi.nlm.nih.gov/pubmed/4640371 However, that...

09 October 2017 7,784 2 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

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

Do you kow of any database containing data on retention time, log P and polarizability for a large number of molecules?

Hi, I am a chemistry undergraduate doing my dissertation project on HPLC. In my project, I need to predict the HPLC retention time of 20 aromatic molecules, the calibration set. To do this I will...

24 February 2021 2,602 1 View

Any easy method of analyzing vitamin D in foods by HPLC?

Looking for method of extracting and analyzing vitamin D in foods by HPLC

24 February 2021 7,524 7 View

What HPLC Marker(s) should be used to detect anthocyanins in a sample?

What HPLC Marker(s) or standard is used to detect and quantify anthocyanins such as Delphinidin, Cyanidin in a given sample of plant extract?

23 February 2021 1,832 4 View

HPLC baseline failure (ACN/Water)?

My baseline increases without any explanation, it is only fixed by changing the solution B, will it be the column? Reagents? Bomb? Detector? Solution A: H3PO4 0.01% IN WATER Solution B: H3PO4...

21 February 2021 5,656 3 View

Which vial septa the right one to be used for HPLC?

This is the unanswered question stored very long in my mind and till now I could not find a right answer. Google search educated me that the red surface is the PTFE material compatible to wider...

21 February 2021 546 3 View

What is the stability of 2 3-dihydroxyquinoxaline in acidic water?

I'm developing a UHPLC assay, and the fluorescent product is 2,3-dihydroxyquinoxaline. The reaction to produce this requires hydrochloric acid (about 1.2 M). Will storage in this acidic...

20 February 2021 5,820 4 View