Gary Robertson

14 Questions 13 Answers 0 Followers

Questions related from Gary Robertson

What should my PVDF membrane look like post transfer during a Western Blot?

When I perform western blot experiments I often get inconsistent results post transfer. What I mean is that when I look at my PVDF membrane after the transfer step I can often see the molecular...

09 September 2018 4,880 4 View

Why does my plasmid run heavy on an Agarose gel?

To test if my double digest of the pTriex-3 vector is working I ran my 'uncut plasmid', 'double digested plasmid' and 'enzyme 1 cut plasmid' and 'enzyme 2 cut plasmid' on an agarose gel. Double...

03 March 2018 4,249 2 View

How did my insert vanish from my plasmid?

Short version: My plasmid contained insert but later after a second transformation into a second cell line the insert vanished and does not show up on PCR or sequencing Long version: I used...

02 February 2018 6,707 9 View

Have my BL21 cells gained antibiotic resistance?

My BL21 cells when grown on an Ampicillin agar plate, completely cover the plate in a lawn as fast as 5 hours. They do this regardless of whether I transform them or not. A plate treated with no...

02 February 2018 361 5 View

How can I delete a large DNA fragment from my gene of interest using PCR techniques?

I have a gene of interest approximately 2000 bp long coding for a protein approx. 670 amino acids in length. I wish to excise one of the proteins domains from the gene. To do so I need to remove...

09 September 2017 2,055 6 View

What is the detection limit for a species (in time) of absorbance spectroscopy?

How long must a species last for, for it be detectable through typical absorbance spectroscopy. Say I have an enzyme with a turnover number of 300. Each reaction would take only 3.3 milli seconds....

08 August 2016 1,133 4 View

Do cumulitive effects on specific activity indicate a concerted mechanism?

I am looking at an enzymes reaction mechanism. I have investigated 2 mutations. both mutations individually reduce the enzymes catalytic activity. The 2 mutations combined render any product...

04 April 2016 2,745 10 View

Can you relate the energy of enzymatic steps to Kcat?

If I know that the free energy barrier of the first step of a enzymatic reaction has increased (And I know by how much) but the other steps remain unchanged may I infer from this any kind of...

01 January 2016 3,396 7 View

How to determine the pKa of Glutathione (GSH) bound to proteins?

https://www.ncbi.nlm.nih.gov/pubmed/2742854I am following the method of Graminski in the paper above. Whether GSH is protonated or not may be followed with absorbance spectroscopy at 239 nm. To...

01 January 2016 3,779 4 View

Can anyone help with multiple bands after DNA double digest?

I am doing an Nde1 and BamH1 double digest. The gel is as followed Pet-28a with no insert (undigested), pet-28a (digested),Thermoscientific zip ruler express 2, pet-11a with insert (undigested),...

07 July 2014 485 7 View

Can anyone help with problems with non linear elution pH gradients during Chromatofocusing?

I am using GE healthcare poly-buffer 96 and PBE 94. I use a 0.025 M ethanolamine start buffer pH 9.4 and an elution buffer of PB 96 pH 6.0. I take care to degas all my buffers, keep the...

07 July 2014 2,721 2 View

Any suggestions on analyzing protein-protein interfaces - angles between subunits?

I would like to compare two homo-dimers. Both Share one identical subunit (which I have aligned structurally) which binds to a second subunit. The specific piece of data I would like to compare is...

05 May 2014 5,727 6 View

Can anyone suggest a program for analysing protein-protein interfaces (ASA, Planarity, Gap volume index, Residues etc.)?

Can someone please suggest a good programme that will either run on-line through a server on on windows that can calculate the ASA, planarity and Gap volume Index of a protein interface. I would...

05 May 2014 6,985 4 View

How to create a diagram/schematic of protein-protein interfaces?

I need a way to create a simple to understand but informative diagram or schematic of a protein-protein interface. I am aware of the program Dimplot but would prefer to try something else. Any...

04 April 2014 5,855 3 View