06 June 2019 2 7K Report

Hi all,

Does anyone have any experience using papain digests of 3D scaffolds followed by Hoechst staining? It is one of the more popular methods I've seen in literature especially for collagen-based scaffolds. I have just completed my own work and the DNA amounts are surprisingly low at 70-100ng/scaffold post 7 days of culture!

I used the general parameters I've seen by most; 125ug/mL Papain in its suitable activation buffer, 60 degrees, 18 hours.

Alongside this I ran an alamarBlue (AB) assay prior to the digest - the results from this would suggest I had my best cell seeding efficiency to date as I had significantly increased reduction of AB at Day 1 vs. previous seeding procedures. So I'm not sure why I'm achieving such low DNA amounts vs. similar attempts by others who achieve 1-2ug +/scaffold after several days culture.

Some papers have used 24 hour digestion periods so perhaps that will assists but if anyone has any experience let me know.

Kind Regards,

John.

Similar questions and discussions
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

How can I assign a velocity along one direction to CO2 molecules in a molecular dynamics simulation using GROMACS?

Dear all, I am trying to perform a MD simulation about the interaction between CO2 and a polymer. I create my model, solvate it and perform the NVT and NPT equilibration. Now I am not able to...

26 February 2021 10,014 3 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

Recalcitrant seed short-mid storage method?

Dear peer scientists, Ultradry storage is not suitable for recalcitrant seeds. However, theoretically, if we can stabilize the structure of the subcellular membrane, as well as glassify the...

25 February 2021 2,442 1 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

What software programs can directly simulate Additive Manufacturing Directed Energy Deposition (DED)?

I understand there are a lot of software programs that can simulate DED by going through specific modeling, simulation, and commands. For me, that was time consuming and results were not accurate....

24 February 2021 4,428 3 View

Our partners will collect data and use cookies for ad personalization and measurement. Learn how we and our ad partner Google, collect and use data. Agree & close