Services directly from the manufacturer

Davids Biotechnologie

How to prepare your cells for sending

How to send cells

1. Sending frozen on dry ice.
a) Frozen in 5-10% DMSO in Medium with 10-20% Serum or serum free if special media were used.
b) Frozen in 5 % glycerin in Medium with 10-20% serum or serum free if special media were used.

2. Sending in culture.

a) Centrifuge cells in log phase from a 25 cm2 flask in a 15 ml tube at 300 x g for 5 minutes.
b) Discart supernatant and add 13 ml of fresh cell culture medium which is used before for culturing your cells.
c) Close the tube well and send together with a non frozen ice pack.   This methods works for sending up to 7 days depending on cell line.

Methods

How to prepare your antigens for sending

How to send the protein antigens?
1. If your antigens are stable at 4 Celsius you can send them on water ice or on dry ice.
2. If your antigen is not stable at 4 Celsius or if you are not shure you can send the antigens best in lyophilized form or on dry ice.
3. Most proteins are also very stable if they are stored and send as a 80% saturated ammonium sulphate precipitate (560 mg/ml ammoniumsulfate).
4. If your protein is within a acrylamide gel matrix you can stain it with coomassie staining and destain it with 3% acetic acid 12% ethanol. Then cut the stained target protein and put it into a eppendorf tube or an other vial without the addition of any solution.

 

Click here for direct contact:

Info@dabio.de

Methods Collection

Standard affinity purification

1. Washing of matrix.

2. Elution buffer 2 CV

3. Equilibration with PBS

4. Binding of Serum or IgG fraction. All fractions must be filtered with 0,22 µm and buffered to pH 7.

5. Washing with PBS

6. Elution with pH 2,8 buffer.

7. Directly buffering the eluted IgG to get pH 7

8. Washing of matrix and start with step 3 for more runs or store at 4 Celsius after addition of a preservative.

Please make also use of our affinity purification services. Ask for our training courses for affinity purifications. Our standard affinity purification matrices can be done via Cystein, via n-terminus, via c-terminus, via Carboxyl, via Hydroxyl, via Carbonyl groups in your antigen (protein, peptide, other molecules).

GST tagged proteins for Antibody development and how to prepare the resulting antibodies

1. Using the GST tagged protein and use the resultung antisera directly. This is possible if the anti-GST antibodies do not disturb your assays.

2. Cleavage of the GST tag from your protein with a protease and use the protein without the GST tag for the immunizations. Sometimes there can be loss of protein and we recomend this method only if there is enough protein availlable.
We offer this service for EURO 233

3. Depletion of anti-GST antibodies from your antiserum with a GST-tag affinity matrix. We offer this service for EURO 144. This method is working very well.

4. Depletion of anti-GST antibodies from an affinity purified IgG fraction purified against the GST-tagged protein. We offer this service for EURO 144. This method is working very well.

5. Doing an affinity purification with the protein with a different tag (example HIS– tag) bound to an affinity chromatography matirx. We offer this servie for EURO 144. This method is working very well.

About storage of your antibodies

Serum can be stored from +4^oC with preservatives or frozen –18 to –30^oC

 

Purifed antibody fractions needs sometimes stabilizers to prevent polymerisation.

You may add Arginine or Histidine or Glycine in concentrations of 10 to 200 mM.

Or purified serum albumine at up to 1 % endconcenttration.

 

Monoclonal antibodies

Avoid freezing/thawing cycles as this will reduce the antibody activity about 10 % each cycle.

 

Preservations:

a)  0,1% Sodium Azid (add new every 12 months)
b)  Thymol saturated. You can add some cristals that do not dilute.

 

 

Elisa Standard Method with alkaline phosphatase detecting secondary antibody. This method is useful to repeat our standard elisa used for titre detection of the antisera.

 

Coating with antigen.
100 ng/ 100µl in each well. Diluted in 0,1M bicarbonate pH 9,4.
Incubation over night at 4 Celsius

Blocking with unrelated protein mix
200 µl blocking solution (Cat.No D303-50))
Incubation 30 minutes at room temperature.

Washing with each 250 µl.
3 x water PBS.   
1 x incubation solution (Cat.No. D302-50)
3 x water PBS

Incubation with 1. Antibody.
Different dulutions.
Dilutens in incubation solution (Cat.No. D302-50)
Incubation over night at 4 Celsius or at room temperature for one to 8 hours.

Washing with each 250 µl.
3 x water PBS.   
1 x incubation solution (Cat.No. D302-50)
3 x water PBS

Incubation 2. Antibody conjugated to alkaline phosphatase
Earlier optimized concentration of 2. antibody
Incubation at room temperature for 3 hours or over night at 4 Celsius.

Washing with each 250 µl.
3 x water PBS.   
1 x incubation solution (Cat.No. D302-50)
3 x water PBS

Enzyme color reaction.
1 mg PNPP in 1% Diethanolamin pH 9.8
Incubation at room temperature for 3 to 6 hours or 16 to 24 hours at 4 Celsius

 

For blocking you may also use 5% BSA in PBS
For incubation you may also use 1 % BSA in PBS with 0,05% Tween 20 giving in some cases lower signals.

Preparing proteins from page gels

for Electroeluting
Acrylamid gel method for electroelution
1. The gels are fixed with 50% Ethanol/2% acetic acid.
2. Staining with Coomassie R250 gel staining. Coomassie dye in 25% Ethanol/5% acetic acid.

3. Destaining is with 10% Ethanol/1% acetic acid.

4. Cut the band from the gel.

5. Put the gel pieces into a tube without adding any solution.

6. Send the tube with the protein in the gel into our lab.

 

 

Coomassie staining solution specifically designed for later electroelution  can be ordered together with a protocol at Davids Biotechnologie.

100 ml Cat.No. P601 € 55