Lysing membrane receptors for pulldown?

12 December 2018 1 6K Report

Dear all,

we are trying to change our lysis method for membrane receptors (using Molon buffer, 15min ice, 15min centrifugation 14'000g, then overnight on beads) with receptor-HA constructs in mammalian cells and HA-beads, to receptor-StrepTagII constructs, to get (hopefully) more purity and higher yield.

However, some reagents in our buffer aren't that compatible with the StrepTagII system, therefore I want to ask if you have any working system for membrane receptor pulldown using the StrepTagII system?

Best, Chris

Christoph Matti

In case anyone finds this question, we succeded in using CHS, DDM as common, and then simply diluted by a factor of 10 to end up with 0.1% DDM which also then worked with Streptactin. :-)

Badges
Science method

More Christoph Matti's questions See All

Protein expression decreases in bacteria over time(months)?

We are experiencing a decrease in protein expression in our BL21 DE3 strain. So far we were able to rule out an influence of i.e. IPTG (prepared new fresh stock), however we have no clue what else...

07 August 2019 959 3 View

C4, C8 or C18 Column for protein polishing step?

Dear all, I want to purify a final tagging reaction, where a 12kDa protein gets a chemical 1.5kDa tag via enzymatic method by a 60kDa transferase. As it is quite sticky, I don't want to use FPLC...

08 September 2017 7,424 7 View

Comparing different qPCR amplicons, how to account for different length of amplicons when using SyberGreen?

Dear all, I performed several qPCR, which also worked fine, however, now during the evaluation I encountered an issue. My problem is that due to the restriction when choosing primers, I ended up...

07 August 2014 8,822 3 View

For ELISA, which capture/coating AB concentration should I use?

I am developping an ELISA, which seems to work, although I am puzzled by the titration of the capture AB. The Assay: All samples were in dublicates, volume used 50ul, except 150 for blocking,...

03 April 2014 167 8 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

How can you bring two structures together to create an adhesive bond in LAMMPS?

I have created an Ice 1h crystalline structure and an Aluminium substrate structure and equilibrated both at 250K. Now I need to bring them together in a way that an adhesive bond is created....

01 March 2021 3,325 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

Necessary tube lenght to cool Natural gas in a dry-ice cooling bath?

I have a tank which contains natural gas at 250 Bar and 293 Kelvin. I want to cool the gas in a dry-ice cooling bath to about 195 Kelvin. The gas will flow in a copper-tube through the bath with a...

27 February 2021 8,128 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