Sir/Madam please suggest me a proper protocol?

More Srikanth Konde's questions See All

Does the urea contain conductivity?

Solubilization buffer 2M Urea, 1mM DTT, 1mM EDTA & 10mM Tris.

31 December 2016 3,855 4 View

Is there any possibility to give negative results in ELISA? If my protein present in Urea solution

My protein present in 8M Urea, 1mM DTT, 1mM EDTA, 1mM PMSF, 10mM Nacl & 50mM Tris @pH 8.0 And it is buffer exchanged with 50mM Tris, 10mM Nacl, 1mM DTT,EDTA & PMSF @pH 8.0

31 December 2016 4,460 3 View

Is there any possibility of variation in PI value of a protein with theoritical value and actual value?

my protein is cry1 ac mol.wt 133.33 but it is precipitating below pH value @8.0

31 December 2016 3,490 3 View

How can i purify my protein?

my protein PI is 5.0, with no Tag and mol.wt is 133kDa

11 December 2016 4,175 0 View

Is there any possibility of degradation of protein in 2M Urea?

My protein is recombinant one

11 December 2016 3,269 7 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

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

How to improve the buffer capacity of sea water?

Hello everyone I would like to improve the buffer capacity of seawater (pH ~8) in order to dissolve as much CO2 as possible without dropping the pH below 7.5 (a range between 7 - 7.5 is...

17 February 2021 2,797 2 View

Why does flow deformation occur at the test line and control line on nitrocellulose membranes (LFIA)?

I use 1% BSA in 10 mM phosphate buffer (pH 7.4) solution when striping my nitrocellulose membranes with antigens and antibodies. However, I always observe deformed flow when my sample or running...

16 February 2021 4,224 3 View

Omar Gonzalez-Ortega

You will find everything you need here

http://wolfson.huji.ac.il/purification/

Srikanth Konde

thank you sir Omar Gonzalez-Ortega

Steingrimur Stefansson

Hi Srikanth, alternatively you can harvest the E. coli before your protein gets deposited into inclusion bodies.

The yields will be lower, but the protein will still be soluble in the bugs and you don't need to deal with denaturation/renaturation of inclusion bodies that might or might not work.

Steingrimur Stefansson thank you for your good suggestion