ࢿૡቧᇦ 162-0239 Immun-Blot PVDF/Filter Paper Sandwiches, 8.5x13.

5cm, 50 Imaging and Electrophoresis

Cat number Description pack
Standards 170-3966 Extra Thick Blot Paper, 7x8.4cm for mini-gels, 60 sheets
170-3967 Extra Thick Blot Paper, 8x13.5cm for criterion-gels, 60 sheets
161-0363 Precision Plus Protein Unstained Standards, 1ml, 100
170-5070 Immun-Star WesternC Chemiluminescence Kit, includes 50ml of
applications
luminol/enhancer, 50ml of stable peroxide buffer
161-0373 Precision Plus Protein All Blue Standards, 500̂l, 50 applications
ဧิࡼWestern Blotဣዩৎଝ౐ႥĂᓰཀྵ 161-0374 Precision Plus Protein Dual Color Standards, 500̂l, 50 Electrophoresis and Blotting Tanks
applications 165-8001 Mini-PROTEAN Tetra Cell, 10-well, 1.0mm thickness, 4-gel
ဣዩഗ߈ ၄ਐୢ፿ઓ Ꮎᒜୢ፿ઓ Bio-Rad౐Ⴅဣዩഗ߈ ଥᒋჅᏴ 161-0377 Precision Plus Protein Dual Xtra Standards, 500̂l, 50 system includes 5 combs,
applications 5 sets of glass plates, 2 casting stands, 4 casting frames, sample
ืୢᓰ۸ 2ࡵ16ቃဟ Ready GelᎾᒜୢ૞ TGX Stain-FreeTMᎾᒜୢ ᇄኊဧ፿஠ቲreprobingࡼఝᄏLj 161-0385 Precision Plus Protein WesternC Pack, includes 250̂l of loading guide, electrode assembly, companion running module,
CriterionᎾᒜୢ ᑚ‫ݝ‬ॊஂဏࡼॅ፿భጲ৪൰35్ Precision Plus Protein WesternC Standards and 125̂l of tank, lid with power cables and mini cell buffer dam
ቃቯᎾᒜୢ૞ᑗ22్ᒦቯᎾᒜୢ StrepTactin-HRP Conjugate, 50 applications 170-3935 Mini Trans-Blot Module includes 2 gel holder cassettes, 4 foam
DŽభᏥቲᔢࣶ572ৈዹອDž Buffers, Reagents and Stains pads, modular electrode assembly, blue cooling unit
165-6001 Criterion Cell, includes electrophoresis buffer tank, lid with power
161-0737 Laemmli Sample Buffer, 30ml
࢟፺ 1ቃဟ 1ቃဟ 15ॊᒩ ஂᏖဟମ cables, 3 sample loading guides
161-0710 2-Mercaptoethanol, 25ml
170-4070 Criterion Blotter with Plate Electrodes, includes cell assembled
161-0732 10x Tris/Glycine/SDS Buffer, 1L
GelDoc EZ for ᇄࠥၫ௣ ᇄࠥၫ௣ 2.5ࡵ5ॊᒩ 1. ᇄኊဧ፿ఝᄏࡼᑞဣ with plate electrodes, lid with cables, 2 Criterion gel holder
161-0771 10x Tris/Glycine/SDS Buffer, 5L
cassettes, 1 pack precut blot absorbant filter paper, 4 foam pads,
Stain-free imaging Loading Control 161-0734 10x Tris/Glycine Buffer, 1L
gel/blot assembly tray, roller and sealed ice brick
2. భጲऱ‫ࡼܣ‬ᎁછࡨ‫ڹ‬ᒠᓞ፝ᄟୈ 161-0771 10x Tris/Glycine Buffer, 5L
170-4071 Criterion Blotter with Wire Electrodes, includes cell assembled
3. ࡨ‫ڹ‬ᒠዹອᒠ೟఼ᒜ 161-0780 10x Phosphate Buffered Saline, 1L
with wire electrodes, lid with cables, 2 Criterion gel holder
4. GelDocEZ ߅ ስ ᇹ ᄻ భ ஠ ቲ 170-6435 10x Tris Buffered Saline, 1L
cassettes, 1 pack precut blot absorbant filter paper, 4 foam pads,
ᔃᅪĂ‫ڹ‬਒Ă౸਒ਜ਼Stain-Free 170-6404 Blotting-Grade Blocker, nonfat dry milk, 300g
gel/blot assembly tray, roller and sealed ice brick
161-0786 Bio-Safe Coomassie Stain, 1L
ࡼࣶᒬ።፿ဣዩ 170-3940 Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell, includes
161-0495 Oriole Fluorescent Gel Stain, 1x solution, 200ml
Trans-Blot SD transfer cell, trans-Blot SD agarose gel support
161-0491 Flamingo Fluorescent Gel Stain, 10x solution, 100ml
frame and extra thick blot paper
Precast Gels 164-5052 PowerPac HC Power Supply
ࡨ‫ڹ‬ᓞ፝ 456-903x Mini-PROTEAN TGX Precast Gel, Any kD Resolving Gel,
1ࡵ16ቃဟ 1ࡵ16ቃဟ 3ࡵ15ॊᒩ ஂᏖဟମ Imaging Systems
10 gels/box
170-8270 Gel Doc EZ System, includes darkroom, camera, cables and
456-109x Mini-PROTEAN TGX Precast Gel, 4-20% Resolving Gel,
ဧ፿඾ጽजଶ‫ހ‬෹‫ܪ‬ Ꮦ5ቃဟ Ꮦ5ቃဟ Ꮦ5ቃဟ Image Lab Software
10 gels/box
ࡨ‫ڹ‬ᒠ 170-8271 UV Sample Tray for gels using ultraviolet illumination
456-108x Mini-PROTEAN TGX Precast Gel, 4-15% Resolving Gel,
170-8272 White Light Sample Tray for gels stained with Coomassie Blue,
10 gels/box
߅ስਜ਼ၫ௣ॊᇜ ဧ፿Filmᄟୈሆ ဧ፿ChemiDocXRS+ ဧ፿VersaDocᇹᄻ5ࡵ10 1. ஂᏖဟମ 456-812x Mini-PROTEAN TGX Stain-Free Precast Gel, Any kD Resolving
copper, silver or zinc stains
0.5ࡵ1ቃဟ ᇹᄻ5ࡵ10ॊᒩ ॊᒩ 2. ஂᏖॅ፿DŽ፼ܰ೫FilmDž Gel, 10 gels/box
170-8273 Blue Light Sample Tray for gels stained with GelGreen or any
3. ણஹᎍੑDŽᇄᇁ཰ृ໰ᇕDž SYBR stain
456-809x Mini-PROTEAN TGX Stain-Free Precast Gel, 4-20% Resolving
170-8274 Stain-Free Sample Tray for stain-free gels and blots
4. ᇄኊ‫ڗ‬၀ Gel, 10 gels/box
170-8650 Molecular Imager VersaDoc MP 5000 System, includes
456-808x Mini-PROTEAN TGX Stain-Free Precast Gel, 4-15% Resolving
Gel, 10 gels/box
CCD camera, darkroom, power supply, cables, epi-illuminator,
transilluminator, fluorescence reference plate, focusing target and
Fast Western Blot Workflow ౐Ⴅᓞ፝ဣዩഗ߈
ဧ፿Re-probingࡼऱज Ꮦ 5 ቃဟLj৪൰ Ꮦ5ቃဟLj৪൰ఝᄏ ᇄኊဧ፿Re-probe஠ቲ 1. ஂᏖॅ፿ 567-112x Criterion TGX Precast Gel, Any kD Resolving Gel, 1 gel/box
஠ቲLoading Control ఝ ᄏ ॅ ፿ Ꮦ ॅ፿Ꮦ350ගᏄ Loading Control DŽᇄኊ৪൰reprobingࡼఝᄏDž 567-109x
567-108x
Criterion TGX Precast Gel, 4-20% Resolving Gel, 1 gel/box
Criterion TGX Precast Gel, 4-15% Resolving Gel, 1 gel/box
170-8265
Quantity One Software
Molecular Imager ChemiDoc XRS+ System with Image Lab Superior and More Reliable
350ගᏄ 2. ஂᏖဟମDŽᇄኊreprobingDž Software, includes darkroom, UV transilluminator, epi-white
567-812x Criterion TGX Stain-Free Precast Gel, Any kD Resolving Gel,
3. ᇄኊဧ፿഍ጙ్ୢ౶஠ቲ 1 gel/box
illumination, camera, power supply, cables and Image Lab
Loading Control Software
567-809x Criterion TGX Stain-Free Precast Gel, 4-20% Resolving Gel,
4. ‫્ݙ‬Ᏻ߲ሚLoading Controlࡼ 1 gel/box
ࡨ‫ڹ‬ᒠ࿟ዹ೟ਭࡍࡼ༽ౚ 567-808x Criterion TGX Stain-Free Precast Gel, 4-15% Resolving Gel,
5. ဧ፿ᑞဣࡼTotal Protein
Loading Control
1 gel/box
Blotting Membranes and Immunodetection
SYBR is a trademark of Invitrogen Corporation.
NOTICE TO PURCHASER: LIMITED LICENSE
ීᇠࡼLjણۣࡼLjᎌ቉ࡼ
162-0112 Nitrocellulose Membrane, 0.2̂m, 30cm x 3.5m roll * This product is sold under license from Life Technologies Corporation, Carlsbad CA
ቃஉ Ꮦ15ቃဟ Ꮦ12ቃဟ Ꮦ6ቃဟ 162-0213 Nitrocellulose/Filter Paper Sandwiches, 0.2̂m, 7x8.4cm, 50 pack for use only by the buyer of the product. The buyer is not authorized to sell or resell
162-0233 Nitrocellulose/Filter Paper Sandwiches, 0.2̂m, 8.5x13.5cm, 50 pack this product or its components Bio-Radࡼᓜಽଆၣ‫ۑ‬ᓐิଝႥᓞ፝DŽWestern BlotDžဣዩഗ߈ǖ
162-0115 Nitrocellulose Membrane, 0.45̂m, 30cm x 3.5m roll
162-0215 Nitrocellulose/Filter Paper Sandwiches, 0.45̂m, 7x8.4cm, 50 pack ■ 1ቃဟดᅲ߅࢟፺ਜ਼ᓞ፝
162-0235 Nitrocellulose/Filter Paper Sandwiches, 0.45̂m, 8.5x13.5cm, 50 pack
162-0177 Immun-Blot PVDF Membrane, 26cm x 3.3m roll ■ ᑞဣࡼLoading Control
162-0219 Immun-Blot PVDF/Filter Paper Sandwiches, 7x8.4cm, 50 pack
■ ࡨ‫ڹ‬ᒠዹອᒠ೟఼ᒜ
‫݌‬ಘညෘጛኧ‫ޘ‬ອ(࿟਱)ᎌሢ৛ႊ ۱ய‫ࠀူێ‬ ਓᒳ‫ࠀူێ‬ ᇊੈ‫ࠀူێ‬
࢐ᒍǖ࿟਱ှ໌ࣁቤཌࣁऱവ!18!੓ ࢐ᒍǖ۱யޫዴཌၝ਒ᇝಱ!5!੓ ࢐ᒍǖਓᒳှણှࣁവ!403!੓ ࢐ᒍǖઌ۱ဏᇊੈှੈాཌஊहࡍࡸ!634!੓
ۣಽਓ‫!ޝ‬E!ࣆ!3!ണ A!ࣆॣષᒙ࢐ਓ‫!ޝ‬22!ണ ਪଔ࢟ᔇࡍሇ!1302-03!၀ ቤီஏᒦቦ!B!ᔭ!1202!၀ ■ ᇄኊဧ፿ActinĂTubulin૞GAPDH஠ቲreprobing
࢟જǖ021-6169 8500 ࢟જǖ010-5939 0088 ࢟જǖ020-8732 2339 ࢟જǖ027-8380 6255
ࠅᑞǖ021-6169 8599 ࠅᑞǖ010-5939 0160 ࠅᑞǖ020-8732 2332 ࠅᑞǖ027-8380 6265
ᎆ‫ܠ‬ǖ200120 ᎆ‫ܠ‬ǖ100028 ᎆ‫ܠ‬ǖ510095 ᎆ‫ܠ‬ǖ430032 ༿षᆰwww.bio-rad.com/geldocez೫ஊᎌਈ
ᒦਪં୸ᒦቦǖ800-820-5567,021-61698504!!!!!ઢ፩षᆰǖwww.bio-rad.com

References Stain Freeଆၣࡼৎࣶቧᇦ

1 http://protocolsonline.com/proteomics/western-blotting/loading-controls/, viewed 22 November 2010.
2 Yperman J, De Visscher G, Holvoet P, Flameng W. Beta-actin cannot be used as a control for gene expression in ovine interstitial cells
derived from heart valves. The Journal of heart valve disease. 2004; 13:848–53.
3 Alexander M, Curtis G, Avruch J, Goodman HM. Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of
glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1985; 260:11978–85
4 Moskowitz PF, Oblinger MM. Transcriptional and post-transcriptional mechanisms regulating neurofilament and tubulin gene expression
during normal development of the rat brain. Brain Res Mol Brain Res. 1995;30:211–22.
5 Dittmer A and Dittmer J. ß-Actin iis not a reliable loading control in western blot analysis. Electrophoresis 2006; 27:2844-2845
6 Aldridge GM, Podrebarac DM, Greenough WT & IJ Weiler. The use of total protein stains as loading controls: an alternative to high- Bulletin 0020 CH Rev A Asia Pacific/China 11-147 0511 Sig 0111
abundance single protein controls in semi-quantitative immunoblotting. J Neurosci Methods. 2008 July 30; 172(2): 250–254.
 ᆐဠඐገ஠ቲLoading ControlǛ ᔪ࡝ጙఘଜࡨ‫ڹ‬Loading Controlࡼ Ŗ ืୢ཰ྻ‫ތ‬ፊLjᄰ‫ޟ‬ణத‫ܟ‬Ꮟࡼ፺ࡸᄟࡒቧ੓ৎ༓ Bio-Radஊ௼ऱ‫ښ‬
ဧ፿Loading Contorlᔫᆐဣዩ࣪ᑍဵWestern Blotဣዩ ဍ଀খ஠ऱ‫ဵښ‬ဠඐǛ Ŗ ဣዩဟኊገᄴဟ๴ೝ్ጙዹࡼืୢLjጙ్፿᎖Total ဧ፿ᓜಽࡼStain FreeืୢభጲయॲTotal Protein Stain
Protein StainLj഍ጙ్፿౶ᔪሆᎊࡼᓞ፝ਜ਼ଶ‫ހ‬ ᒦࡼ৉ᒬ౩ซă჈ᇄኊ཰ྻਜ਼ᅙྻࡼ‫ݛ‬ᒾLjᄋ৙ᆮࢾጙᒘ
ࡼ૥߻Ljጐဵख‫ܭ‬ᎁኅ൙ᆪࡼ૥‫۾‬ገཇăဧ፿˸-actinĂ
ࡼࢅ۳ஶLj‫ܣ‬᎖஠ቲ঱ᒮআቶࡼဣዩă
˸-tubulinਜ਼GAPDHጲૺ໚჈঱‫ࡼࡉܭ‬Đఘଜࡨ‫ڹ‬đᔫ Ŗ ಸࠔ੺཰ྻဵጙᒬTotal Proteinࡼ཰ྻऱ‫ښ‬Ljࡣဵ
1.6
Loading Controlဵ෹༄‫ޟ‬፿ࡼऱजă 1 ᑚᒬऱजᒮআቶਜ਼ጙᒘቶ୷‫ތ‬DŽᄴဟࡀᏴภሶन።Dž ဧ፿Stain Freeଆၣ࣪Western Blot஠ቲTotal Protein
PSD 95
Sypro Ruby Loading ControlဵิࡼᔢଛኡᐋăᑚሲଆၣဧิᇄኊᏳ
Loading Control (LC)ᏴWestern Blotᒦభጲǖ 1.4
■ ᑺဣඛᄟ፺ࡸࡼዹອ࿟ዹ೟ጙᒘ
GAPDH ᄴဟ๴ೝ్ืୢLjᑳৈWBဣዩభጲ౐Ⴅభణࡼᅲ߅ă
DŽ‫ܜ‬඾࿟ዹࡇᇙDžLjᑚᏴ Amido Black

standardized optical density

໚௥ᎌྙሆᄂ࢛ǖ
ኊገ‫ݙ୷܈‬ᄴዹອࡼࡨ‫ࡉܭڹ‬೟‫ތ‬ፊဟᎄᆐᒮገ
1.2
■ ଶ‫ื࠭ހ‬ୢࡵෞࡼᓞ፝ਭ߈ဵ॥቉ൈጙᒘ 1. Stain FreeଆၣጯᎾ࿸ᏴᎾᒜୢดLjᇄኊ཰ྻ‫ݛ‬ᒾ
1 2 3 4 5 6 7
■ ལ࿩ᄂࢾࡨ‫ڹ‬ᄟࡒဟᑺීকዹອ፺ࡸཀྵဣᎌࡨ‫ࡀڹ‬Ᏼ 1.0 2. ࢟፺ઁ5ॊᒩด૾భࡻࡵืୢᅄ
3. ถऱ‫ࡼܣ‬ᎁછᓞ፝ᄟୈ

Faster and More Quantitative ‫ޟ‬፿ࡼLoading ControlᎌࣶభణǛ

ஞဧ፿࡝ጙࡨ‫ڹ‬ᄟࡒᔫᆐLoading Contorlభถ્ࡒ౶ೝৈ
0.8
4. ࠭ืୢࡵෞࡼཝ߈࣒ۣߒ঱ࣞጙᒘቶLjࢅ۳ஶ

౐Ⴅࡼࢾ೟ဣዩഗ߈
0.6
ᆰᄌă 5. ᇄภሶन።DŽ‫ۻ્ݙ‬ᇸᅙDž

■ ᄰ‫ޟ‬ᆸඣ෦ཱྀ঱ॕࣞࡼఘଜࡨ‫ڹ‬Ᏼ‫ݙ‬ᄴ༽ౚሆဵᆒߒੱࢾ 0.4 6. ဧ፿ᄴጙ్ืୢ஠ቲTotal Protein Loading Controlਜ਼

ઁኚࡼWesternဣዩLjᑞᑵᔪࡵጙᒘቶ
Western Blotting ■
೟ࡼLjࡣူဣ݀ऻྙࠥă 2, 3, 4
኏ࣶᆸඣঢቭབࡼࡨ‫ڹ‬ᒠDŽProtein Of Interest, POIDžᎧ
20 25 30
μg total protein
35 40

7. ࡍ७ࣞჁ࣢ဣዩഗ߈Lj6ቃဟดᅲ߅ᑳৈဣዩ
ఘଜࡨ‫ڹ‬ሤ‫ࢅဵ࣒܈‬ॕࣞࡼăPOIᎧLCᄴဟᏴሣቶपᆍด
8. ဧ፿భ၁છࡨ‫ڹ‬ᅄໍ஠ቲዹອᒠ೟ຶৰ
‫ݣ‬ถဧ፿LC࣪POI஠ቲࢾ೟LjဣଔဣዩᒦLjLCསᆁᆁިਭ
Loading Controlဵख‫ܭ‬ጙৈᎌᒠ೟ࡼWestern Blotஉਫ ሣቶपᆍᒄᅪă 5
Correlation Coefficient (r) PSD-95 (POI; n=10) ᅄ2. HeLa ᇼ‫ߥ۝‬ᄋࡨ‫ڹ‬ᒠࡼStain-Freeୢᅄă ဧ፿ImageLabྟୈ
Ⴥ‫ݙ‬భ૞ལࡼ࣪ᑍăBio-Radஉ੝ࣶฤࡼ࢟፺Ăᓞ፝ਜ਼߅ስ PSD-95 (POI; n=10) 0.914 ࣪Stain-Freeୢᅄ஠ቲTotal Protein Loading Controlॊᇜăࡨ‫ڹ‬ᔐ೟ྙ
ဧ፿Total Protein StainᔫᆐLoading Controlభణ൮Ǜ SYPRO Ruby (Total Protein LC; n=11) 0.848 Adj Volăጙ౺ჅာLjၫ௣થመာ೫Stain-Free ଆၣࣖᄂࡼ፺ࡸ۳ஶ
DŽMean Bkgd.DžLj݀ဧ፿ᄴጙ్ืୢ஠ቲઁኚࡼᓞ፝ਜ਼ଶ‫ހ‬ဣዩă
ଆၣᒄࡍ߅Ljᅎ߲ᔢቤࡼStain FreeଆၣLjဣሚ೫ᑳᐽୢࡼ ྙᎎᅄ1ᒦჅာLjဧ፿Total ProteinᔫᆐLoading Control‫܈‬
ဧ፿࡝ጙࡨ‫ڹ‬ᄟࡒৎᆐᓰཀྵă6ࡣဵLjဧ፿ཝࡨ‫ࡀྻ཰ڹ‬Ᏼ
Amido Black (Total Protein LC; n=11)
GAPDH (Single Protein LC; n=10)
0.865
0.253
˸-actin (Single Protein LC; n=6): 0.268
ጲሆ౩ซǖ
Total Loading ControlLjᆐิᄋ৙ৎᆐறᓰࡼဣዩஉਫă ‫ܭ‬1. Stain-Freeୢᅄࡼࢾ೟ॊᇜ
No. Adj Vol. (Int) Mean Bkgd (Int) Area (mm2)
ᅄ1. 10ᐽෞࡼຳ௿ࡨ‫ڹ‬เࣞLjመာ‫ݙ‬ᄴࡼĐሤ࣪ॕࣞđቓൈă SYPRO Ruby 1 5,822,781 258.5 391.8
ਜ਼‫ړ‬૥੨ଶ‫ဵࡼހ‬Total Protein Loading ContorlLj໚Ꭷገଶ‫ࡼހ‬෹‫ڹࡨܪ‬ᒠ 2 6,313,916 254.5 391.8
PSD-95ࡼᑞဣเࣞ୻தLjऎGAPDHਜ਼B-actinᑚಢ঱ॕࣞࡨ‫ڹ‬ᒠࡼLoading 3 5,918,390 250.5 391.8
Controlᐌገ‫ތ‬੪ࣶă6 4 20,307,530 254.0 391.8
5 17,764,777 248.0 391.8
6 17,315,493 242.6 391.8
7 15,999,297 234.7 391.8

Bio-Rad Western Blotting

Key

ࠅᄻWestern Blotဣዩഗ߈
Target protein
Cathode
– Primary antibody
Filter paper

ኊገဧ፿ೝ్ୢLj੒ဟᏖ48ቃဟ Gel
Secondary antibody

Enzyme conjugate
Membrane
Gel 1 Ś Stain with coomassie (1-24 hrs): For loading control Filter paper
Substrate
Product
Anode Emitted light
+

Gel 2 Ś Transfer (60 min) Ś Western blot (3-4 hrs): For sample Prestained Chemiluminescent Fluorescent

ReadyPrep Sample Preparation Kits, Precision Plus ࡨ‫ڹ‬ᒠ‫ܪ‬ᓰອLj๴ୢદߡጘLj Tetra ࢟፺‫ݹ‬ਜ਼ GelDocEZਜ਼ Trans-Blot Turboᓞ፝ᇹᄻLjCriterionᓞ፝‫ݹ‬ਜ਼Mini Trans-Blotᓞ፝‫ݹ‬Lj Immun-StarTM VersaDoc MP 5000
Bio-Rad Stain Freeဣዩഗ߈ Mini and Midi Gels (TGX, HCl, and Criterion࢟፺‫ݹ‬LjPowerPac࢟Ꮞ ImageLabྟୈ Trans-Blot SD‫ۍ‬ছᓞ፝‫ݹ‬LjᎾ૘ࡼᓞ፝દߡጘਜ਼Ꮎᒜࡼᓞ፝፿ෞ WesternCછኧख਒ ChemiDocXRS+
ဧ፿ጙ్ୢ᎖6ቃဟด૝ࡻৎଝறཀྵࡼဣዩஉਫ Stain-Free), Sample Buffer ଶ‫ހ‬၂ଋਫ਼
Only 1 Gel Ś Imaging (2-3 min) Ś Transfer (3-15 min) Ś Western blot detection (3-4 hrs)
Sample and Gel Preparation Protein Electrophoresis Analysis Blotting Immunodetection Imaging and
ዹອᒜ۸ਜ਼ืୢᓰ۸ 15 minutes 5 minutes 3-15 minutes 4-5 hours Analysis
ࡨ‫࢟ڹ‬፺15ॊᒩ ߅ስॊᇜ ᓞ፝ਭ߈3-15ॊᒩ ඾ጽଶ‫ހ‬4-5ቃဟ 5 minutes
5ॊᒩ (ॖ‫ܕ‬ĂጙఝĂऔఝ) ߅ስਜ਼ଶ‫ހ‬Lj
5ॊᒩ