Why my Gibson assembly doesn't work?

I'm assembling 3 PCR fragments with my vector pBAD and transforming them into dh5a. The second PCR fragment has 4 variations to it and the template for this PCR fragment comes from a plasmid. I've...

01 February 2019 5,696 3 View

Does anybody know where to purchase anti-H7 for flagellin in escherichia coli?

Im specifically looking for an anti-mouse antibody for flagellin (antisera H7). I've checked papers and googled extensively but couldnt find it. Any ideas?

03 April 2017 2,165 2 View

Does anyone have a good protocol for flagella preparation/purification?

I need a protocol to shear flagella off E.coli O157:H7. I have been using a protocol online that is basically resuspending an overnight culture in a syringe and then, spinning it down to acquire...

02 March 2017 6,637 2 View

How does one crystallise a protein with the presence of TCEP as the reducing agent, and with it's co-factors (NAD+ and Fe2+)?

I have a protein which I know is stable with the presence of co-factors (NAD+ and Fe2+). Recently i have been trying to purify it with the TCEP as a reducing agent since my protein is known to...

10 November 2016 185 2 View

Why mbp-cleaved protein disappears after anionic exchange?

I am purifying a protein (47 kDa) that is fused onto an MBP (42 kDa). The PIs for both proteins are 6.48 and 4.9 respectively. After purifying it using a dextrose column, I have buckets of fused...

04 May 2016 8,945 8 View

How to find self-associating motifs in a protein sequence?

04 May 2016 3,195 3 View

Why did the cleaving of my MBP tag on my protein failed?

I'm purifying a protein that is fused onto an MBP tag (42.5 kDa). My protein size is roughly the similar size of the tag (47kDa). Together, they form a complex about 89 kDa. After a run through a...

03 April 2016 8,667 3 View

How do I know if a flagella is in R or L conformation?

I have sequenced my sample and found some mutaions in the fliC gene. I have found that they have introduced some aa changes however, I would like to know if this changes the conformation into R or...

02 March 2016 7,471 1 View

How to generate GNOM file (.out) for DAMMIF runs?

I have .dat files from scatter that contain the Dmax of a protein and now I want to generate ab initio models. however I dont know how to generate .out files that are needed byy DAMMIF programs on...

09 October 2015 9,090 1 View

Why does the pressure alarm keeps going off during Nickel column purification?

I'm using a new column (5 ml Ni NTA HiFliQ column from Generon) to purify my protein AdhE which is Histagged and sized 96 kDa. However, during purification, the alarm pressure keeps going off even...

07 August 2015 5,183 10 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

Benefits of the 24 welled plate?

We have always used the 96 welled plate, the 48 welled plate but apparently there is much evidence on the 24 welled plate and above all its benefits.It states it is the same you would use on ELISA...

28 February 2021 1,149 1 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

In methylene blue test Why is UV absorbance reading very low as soon adsorbent is added and then increasing before reaching equilibrium?

Hello, I'm working on removal of methylene blue. 01. in the dark test very first reading is taken 15 minutes after adding adsorbent to the dye solution and that UV absorbance reading is very...

19 February 2021 472 5 View