• Learn operation of Electrophoresis tank and Electrophoreis Power supply.
• Read following documents

Electrophoresis is a comprehensive term that refers to the migration of charged solutes or particles of any size in a liquid medium under influence of an electrical field.

• Document and revise common laboratory informations related to the examinations

1. Whole blood sent in EDTA vacuttee is acceptable.
2. If required the procedure can be performed with clot of plain sample or flouride sample.
3. Perform the procedure within a week preferably on thursday or friday.
   1. Complete analysis within the evening of run.
   2. Do not wash samples till day of analysis, because RBC gets nutrients from plasma. They are better preserved in whole blood.
4. If RBC have settled completely, remove supernatant plasma
5. If RBC have not settled completely, centrifuge 3000 rpm for 10 minutes before removing supernatant.

1. Prepare wash solution containing 10 mmol/L EDTA and 5% Glucose. Dextrose Wash Solution Preparation

1. Add Dextrose Wash Solution upto 4.0 ml in EDTA vacuttee.
2. Centrifuge the EDTA vacuttee at 3000rpm for 10 minutes.
3. Remove saline part from the vacuttee.

1. First wash
   1. Add Normal saline upto 4.0 ml in EDTA vacuttee.
   2. Allow thoroughly mixing of Normal saline & cells parts of sample By inverting a few times.
   3. Centrifuge the EDTA vacuttee at 3000rpm for 10 minutes.
   4. Remove saline part from the vacuttee.
2. Second wash
   1. as above
   2. 
3. Prepare a set of eppendroff cups with sample_id label to match the sample batch.
4. Take 100 ul of Hb A Hb F Hb S control(s).Take 100 microliter of control & tests samples in eppendroff cups.Add 100ul Haemolysate solution in each cups for hemolysis.Mix it properly.Make sure that sample becomes completely clear.
5. Half diluted TEB buffer

1. The surface of the slide on which gel has to be applied, is made rough using a glass paper.This is done for proper fixation of the gel on the slide.
2. Before applying the gel on the slide wash 2 slides(Non siliconized slideSiliconization of Glassware on which gel will be applied and siliconized slide will be used as a slider) and dry it with tissue roll.
3. Place 2 slides on a levelled surface as shown in above figure.Cut a rectangle of slide size from a x-ray plate, cut a four boardered frame from a x-ray plate,so the thickness of gel is same as thickness of x-ray platecurrent slide

1. prepare 1% Agarose with TEB buffer
2. Then pour hot agarose gel on non siliconized slide carefully.Make a wedge of siliconized slide on non siliconized slide containing agarose gel(as shown in figure).
3. 
4. Carefully press the non siliconized slide over the siliconized slide,so that air do not get trapped in between two slides.
5. Gel will be ready to use after 10-15minutes.
6. then carefully remove the non siliconised slide just before the sample application and should be away from the fan or heat.

1. Use only freshly prepared slide as slides kept in refrigerator cause significant increase in endosmosis.

OLD METHOD

1. Bring used histopathology blade from histopathology department after permission of in-charge of that respective department.
2. Make 2mm slot at every 5mm distance to prepare comb.
3. Wrap 3 layers of cello tape at each end of applicator comb so that level of applicator comb will remain faction of a millimeter higher from the gel so can prevent through and through cutting of gel and spreading of samples beneath the gel.
4. 
5. Make assembly as shown in photograph below for holding sample applicator over the gel.
6. 

NEW METHOD

1. use the blade(applicator) on which a magnet is attached at the back, fix it temporarily with the scale behind.
2. See proper alignment.
3. place an empty slide beneath the comb.

1. Loading sample over applicator:
   1. the applicator is already attached to the scale by the help of magnet
   2. place 2 Ul hemolysed sample on the root of each comb,
   3. if there are less number samples, then preferably skip the first and last comb,
   4. repeat the sample at least twice.
   5. 
2. SLIDE'S COMB IS FEEDED WITH BLOOD SAMPLES
3. after applying the 2ul sample pull the applicator and wipe off extra blood from behind the applicator with a little piece of tissue paper.
4. Loading sample over Gel:
   1. put prepared gel slide beneath the applicator.

1. The applicator is put over one end of gel loaded slide away from margin.
2. Wait for 2-3 minutes,allow the samples to get completely absorbed within gel otherwise samples will spread.
3. Then remove the applicator carefully.

1. take clean, dry electrophoresis tank
2. fill the both compartment of the tank with TEB buffer with the container till the arrow mark, below separately in each compartment (*same buffer should not be used twice and should be discarded)
3. prepare wick from filter paper. Cut filter paper 7cm (width) X 17 cm (Length) and fold it 1 CM inwards. Wet with TEB buffer. wick should not be used twice and should be discard after one use.
4. After successful sample application,put the gel slide in electrophoresis chamber.
5. Slide should be kept in such a way that the gel is directly in contact with wick.
6. Connect the electrode with the electric box red to red and black to black) of the power supply.
7. Set voltage at 350 to 370 V, ampere 7-15 A and run for 15 to 30 minutes until the bands reach the mid portion.
8. After run,switch off the power supply & remove the slides.

1. Put the sample slide in methanol for 10 minutes for removal of excessive water molecules around proteins.Everytime use fresh methanol.
2. Preheat the Microwave oven or incubator for 5 to 10 minutes.

1. Take away the slide from methanol solution, With a piece of tissue paper,wipe away methanol from the opposite surface of the gel slide .
2. Allow it to dry in pre heated oven after switch off it
3. Do not touch the surface containing gel with samples.
4. Denature protein and fix the gel by heating in pre heated oven.
5. Do not heat the slide constantly as it may get broken down due to excessive heat.
6. Allow the slide to be cool.
7. Remove the X-Ray film carefully without disturbing the gel.
8. Take around 2 ml of a staining solution Amido black stain on the slide and spread it with glass rod on slide so as to stain the proteins over the gel surface.

1. Remove the excess stain by putting in the destaining solution 5% glacial acetic acid for 15 minutes.

1. Allow the slides to dry at room temperature or inside the micro wave.

1. label the slide with appropriate sample id according to sample application.
2. take a picture of the the slide applying a grid, make it black.

1. open gel analysis, segregate the individual sample
2. save each sample in the computer for entering in the data base(cutting of each slides used as attachments)

1. Go to Application menu—–>Click on web browser—–>Open 10.207.3.240
2. Do Login from your login ID.
3. Click on “Edit”—–>Click on “Manage attachment” as shown in below figure.
4. 
5. 2 attachments should be done - 1st is “electrophoresis” and 2nd is “Interpretation” file.
6. 
7. Do 2nd attatchment as shown in below figure.
8. 
9. After click on “Browse”,follow the sequence from where you find the “interpretation file(txt file)”.
10. Application menu—→File manager—→Back up1—→Biochemistry—→Department—→Parameter details—→HBe—→hb interpretation—→select appropriate attatchment.
11. Click on “Edit” and enter relavant sample id as shown in below screenshot.
12. 

dithionate_test_latest.ods

1. Various types of hemoglobins
2. Hb A is moved fastest,then Hb F And Hb S moved very slowly, so its band is near application sites.
3. Correlate result of Hb electrophoresis with Dithionite test.